Methods for distinguishing rice varities

ABSTRACT

Polymorphisms were searched in 24 varieties with large planted acreages in Japan, and the polymorphic sites were compared among the varieties. Thus, polymorphic markers that can be used to distinguish varieties in a simple and quick manner were obtained. The markers showed distinct patterns for each of the varieties, demonstrating that their combination would enable the varieties to be distinguished. Thus, the inventors succeeded in obtaining molecular markers that can distinguish 24 rice varieties. The use of these markers enables closely related rice varieties to be distinguished and identified at the DNA level.

TECHNICAL FIELD

This invention relates to methods for distinguishing between ricevarieties.

BACKGROUND ART

Traditionally, varieties of rice plants or rice have been distinguishedby cultivation traits (e.g., height, number of tillers, days toheading), grain traits before/after polishing (e.g., grain shape,weight, and whiteness), and cooking qualities (e.g., taste). Inaddition, sorting using molecular genetic analysis such as RFLP(restriction fragment length polymorphism) and CAPS (cleaved amplifiedpolymorphic sequence) has become feasible. However, the eyes of anexperienced breeder are required to distinguish varieties by theircultivation traits, and this is not something that just anyone canjudge. In addition, statistical analysis of the traits of unpolished orpolished rice is required, and a certain quantity of rice is required todetermine cooking qualities. Thus, it is impossible to distinguish eachindividual rice grain. In principle, molecular genetic analysis hassolved these problems; however, in fact, while effective fordistinguishing between remotely related varieties, such analysis istroublesome for closely related varieties, because it is difficult toobtain established molecular markers.

By definition, single nucleotide polymorphisms (SNPs) are singlenucleotide differences existing in DNA nucleotide sequences. Inpractice, they often include SSR (simple sequence repeats) and insertionor deletion mutations. It is no exaggeration to say that SNPs cause allgenetic differences detectable using molecular markers such as RFLP andCAPS, and all genetic differences reflected in phenotypes and such. SNPstudies and SNP assay systems have made remarkable progress in recentyears. Currently, an assay system has been developed that allows allsteps, from PCR to a result, to be carried out in a 96-well plate, withno need for electrophoresis, enabling remarkably efficient genotypingcompared to traditional molecular markers.

Recently, the reliability of labeling requirements in the food industryhas become an issue, and rice is no exception. For example, the amountof rice being sold as Koshihikari exceeds the national production ofKoshihikari. Thus, the possibility of false disclosure in the ricemarket cannot be denied, and both consumers and sellers desire assaysthat accurately distinguish polished rice varieties, and determine blendratios.

DISCLOSURE OF THE INVENTION

The present invention has been accomplished by considering the abovecircumstances. An objective of the present invention is to provide novelmethods that enable rice varieties to be quickly and easilydistinguished. More specifically, the present invention aims to providemethods for efficiently distinguishing between rice varieties by usingpolymorphic markers.

In order to solve these problems, the present inventors carried outintensive studies. First, using the rice genomic sequence, primers foramplifying 800 bp to 1 kbp fragments from genomic DNA were designed byselecting mainly putative intergenic regions for those chromosomalregions for which rice genomic nucleotide sequences were publiclyavailable, and by using the sequence of RFLP marker probes and the likefor other regions. The designed primers were used in PCR amplifications,with DNA extracted by a simple method from rice varieties Nipponbare,Koshihikari, Kasalath, Guang-lu-ai 4 (G4, below), Kitaake, and a wildrice (Oryza rufipogon, W1943), as a template, to prepare templates forsequencing reactions. The templates were then subjected to cyclesequencing, and samples for sequencing were prepared. The resultingsequence data was compared between varieties, to search for singlenucleotide substitution polymorphisms. Each variety was sequenced atleast twice with each primer, and only definite cases were deemed to bepolymorphisms.

The nucleotide sequences at positions found to be polymorphic betweenNipponbare and Koshihikari, and between Nipponbare and Kitaake, wereexamined by performing PCR and sequencing as described above, usingtemplates of genomic DNA extracted by a simple method from Nipponbare,Hatsushimo, Mutsuhomare, Yukinosei, Kirara 397, Tsugaruroman,Gohyakumangoku, Morinokumasan, Yumeakari, Hanaechizen, Koshihikari,Tsukinohikari, Akitakomachi, Asanohikari, Aichinokaori, Matsuribare,Hinohikari, Yumetsukushi, Hitomebore, Manamusume, Fusaotome, Dontokoi,Kinuhikari, and Sasanishiki. The nucleotide sequences at the polymorphicsites were compared between the varieties.

Next, primers for detecting SNPs that are useful for distinguishingvarieties were designed, and single nucleotide terminator reactions wereperformed using an AcycloPrime-FP kit (Perkin Elmer) to prepare samplesfor genotyping. Genotyping was performed by using ARVO (Perkin Elmer) tomeasure fluorescence polarization.

The results showed that the markers generated around those positionsdetermined by sequencing to be SNPs displayed distinct patterns, andthat they could be used in combination to sort the varieties intodifferent groups. Thus, the inventors succeeded in obtaining polymorphicmarkers that could be used to distinguish between 24 rice varieties.

As described above, the inventors searched for SNPs in 24 rice varietieswith a large planted acreage in Japan, and obtained polymorphic markersthat enabled the varieties to be distinguished in a quick and simplemanner. They thus accomplished novel methods for distinguishing ricevarieties using the polymorphic markers. The methods of the inventionenable closely related varieties to be distinguished and identified atthe DNA level.

Thus, the present invention relates to novel methods for distinguishingrice varieties in a quick and simple manner, and more specifically, itprovides:

[1] a method of distinguishing between rice varieties, comprising thefollowing steps (a) and (b):

(a) determining the type of a nucleotide at-a position according to anyof the following (1) to (28) in the rice genome, or a nucleotide on thecomplementary strand that composes a base pair with the nucleotide atthe position:

-   -   (1) position 593 in the nucleotide sequence of SEQ ID NO: 1,    -   (2) position 304 in the nucleotide sequence of SEQ ID NO: 2,    -   (3) position 450 in the nucleotide sequence of SEQ ID NO: 3,    -   (4) position 377 in the nucleotide sequence of SEQ ID NO: 4,    -   (5) position 163 in the nucleotide sequence of SEQ ID NO: 5,    -   (6) position 624 in the nucleotide sequence of SEQ ID NO: 6,    -   (7) position 534 in the nucleotide sequence of SEQ ID NO: 7,    -   (8) position 358 in the nucleotide sequence of SEQ ID NO: 8,    -   (9) position 475 in the nucleotide sequence of SEQ ID NO: 9,    -   (10) position 323 in the nucleotide sequence of SEQ ID NO: 10,    -   (11) position 612 in the nucleotide sequence of SEQ ID NO: 11,    -   (12) position 765 in the nucleotide sequence of SEQ ID NO: 12,    -   (13) position 571 in the nucleotide sequence of SEQ ID NO: 13,    -   (14) position 660 in the nucleotide sequence of SEQ ID NO: 14,    -   (15) position 223 in the nucleotide sequence of SEQ ID NO: 15,    -   (16) position 247 in the nucleotide sequence of SEQ ID NO: 16,    -   (17) position 163 in the nucleotide sequence of SEQ ID NO: 17,    -   (18) position 421 in the nucleotide sequence of SEQ ID NO: 18,    -   (19) position 178 in the nucleotide sequence of SEQ ID NO: 19,    -   (20) position 141 in the nucleotide sequence of SEQ ID NO: 20,    -   (21) position 480 in the nucleotide sequence of SEQ ID NO: 21,    -   (22) position 481 in the nucleotide sequence of SEQ ID NO: 22,    -   (23) position 131 in the nucleotide sequence of SEQ ID NO: 23,    -   (24) position 510 in the nucleotide sequence of SEQ ID NO: 24,    -   (25) position 248 in the nucleotide sequence of SEQ ID NO: 25,    -   (26) position 92 in the nucleotide sequence of SEQ ID NO: 26,    -   (27) position 743 in the nucleotide sequence of SEQ ID NO: 27,        and    -   (28) position 552 in the nucleotide sequence of SEQ ID NO: 28,        and

(b) relating the type of the nucleotide determined in step (a) to avariety of rice;

[2] the method of [1], which distinguishes the type of a nucleotide byusing a polymorphic marker characterized by a mutation of any of thefollowing (1) to (28) in the rice genome:

-   -   (1) the nucleotide at position 593 in the nucleotide sequence of        SEQ ID NO: 1 is T,    -   (2) the nucleotide at position 304 in the nucleotide sequence of        SEQ ID NO: 2 is T,    -   (3) the nucleotide at position 450 in the nucleotide sequence of        SEQ ID NO: 3 is A,    -   (4) the nucleotide at position 377 in the nucleotide sequence of        SEQ ID NO: 4 is C,    -   (5) the nucleotide at position 163 in the nucleotide sequence of        SEQ ID NO: 5 is C,    -   (6) the nucleotide at position 624 in the nucleotide sequence of        SEQ ID NO: 6 is C,    -   (7) the nucleotide at position 534 in the nucleotide sequence of        SEQ ID NO: 7 is C,    -   (8) the nucleotide at position 358 in the nucleotide sequence of        SEQ ID NO: 8 is G,    -   (9) the nucleotide at position 475 in the nucleotide sequence of        SEQ ID NO: 9 is G,    -   (10) the nucleotide at position 323 in the nucleotide sequence        of SEQ ID NO: 10 is A,    -   (11) the nucleotide at position 612 in the nucleotide sequence        of SEQ ID NO: 11 is A,    -   (12) the nucleotide at position 765 in the nucleotide sequence        of SEQ ID NO: 12 is T,    -   (13) the nucleotide at position 571 in the nucleotide sequence        of SEQ ID NO: 13 is T,    -   (14) the nucleotide at position 660 in the nucleotide sequence        of SEQ ID NO: 14 is G,    -   (15) the nucleotide at position 223 in the nucleotide sequence        of SEQ ID NO: 15 is A,    -   (16) the nucleotide at position 247 in the nucleotide sequence        of SEQ ID NO: 16 is A,    -   (17) the nucleotide at position 163 in the nucleotide sequence        of SEQ ID NO: 17 is A,    -   (18) the nucleotide at position 421 in the nucleotide sequence        of SEQ ID NO: 18 is C,    -   (19) the nucleotide at position 178 in the nucleotide sequence        of SEQ ID NO: 19 is G,    -   (20) the nucleotide at position 141 in the nucleotide sequence        of SEQ ID NO: 20 is G,    -   (21) the nucleotide at position 480 in the nucleotide sequence        of SEQ ID NO: 21 is C,    -   (22) the nucleotide at position 481 in the nucleotide sequence        of SEQ ID NO: 22 is C,    -   (23) the nucleotide at position 131 in the nucleotide sequence        of SEQ ID NO: 23 is C,    -   (24) the nucleotide at position 510 in the nucleotide sequence        of SEQ ID NO: 24 is A,    -   (25) the nucleotide at position 248 in the nucleotide sequence        of SEQ ID NO: 25 is T,    -   (26) the nucleotide at position 92 in the nucleotide sequence of        SEQ ID NO: 26 is C,    -   (27) the nucleotide at position 743 in the nucleotide sequence        of SEQ ID NO: 27 is G, and    -   (28) the nucleotide at position 552 in the nucleotide sequence        of SEQ ID NO: 28 is T;

[3] the method of [1] or [2], comprising the following steps (a) to (c):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, and

(c) determining the nucleotide sequence of the amplified DNA;

[4] the method of [1] or [2], comprising the following steps (a) to (d):

(a) preparing DNA from a test rice,

(b) digesting the prepared DNA with a restriction enzyme,

(c) fractionating the DNA fragments by size, and

(d) comparing the size of the detected DNA fragment with a control;

[5] the method of [1] or [2], comprising the following steps (a) to (e):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) digesting the amplified DNA with a restriction enzyme,

(d) fractionating the DNA fragments by size, and

(e) comparing the size of the detected DNA fragment with a control;

[6] the method of [1] or [2], comprising the following steps (a) to (e):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) denaturing the amplified DNA into single-stranded DNAs,

(d) fractionating the denatured single-stranded DNA on a non-denaturinggel, and

(e) comparing the mobility of the fractionated single-stranded DNA onthe gel with a control;

[7] the method of [1] or [2], comprising the following steps (a) to (f):

(a) preparing DNA from a test rice,

(b) synthesizing two different oligonucleotide probes labeled with areporter fluorescence dye and quencher fluorescence dye, where anoligonucleotide is complementary to a proximal nucleotide sequencecomprising a nucleotide in a position of any of (1) to (28) of [1], or anucleotide in the complementary strand composing a base pair with thenucleotide at the position,

(c) hybridizing the DNA prepared in step (a) with the probe synthesizedin step (b),

(d) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(e) detecting the emission of reporter fluorescence, and

(f) comparing the emission of reporter fluorescence detected in step (e)with a control;

[8] the method of [1] or [2], comprising the following steps (a) to (h):

(a) preparing DNA from a test rice,

(b) synthesizing a probe in which a sequence complementary to the3′-flanking nucleotide sequence comprising a nucleotide in a position ofany of (1) to (28) of [1], or a nucleotide in the complementary strandcomposing a base pair with the nucleotide at the position, is combinedwith a totally unrelated sequence,

(c) synthesizing a probe that is complementary to the 5′-flanking regioncomprising a nucleotide in a position of any of (1) to (28) of [1], or anucleotide in the complementary strand composing a base pair with thenucleotide at the position,

(d) hybridizing the probe synthesized in step (c) with the DNA preparedin step (a),

(e) digesting the hybridized DNA in step (d) with a single-stranded DNAcleaving enzyme, and dissociating a part of the probe synthesized instep (b),

(f) hybridizing the dissociated probe in step (e) with a probe fordetection,

(g) enzymatically digesting the hybridized DNA in step (f), andmeasuring the fluorescence intensity thus generated, and

(h) comparing the fluorescence intensity measured in step (g) with acontrol;

[9] the method of [1] or [2], comprising the following steps (a) to (f):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) denaturing the amplified DNA into single-stranded DNAs,

(d) separating only one strand from the denatured single-stranded DNAs,

(e) performing an elongation reaction from near a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, whereby the reactionelongates one nucleotide at a time, then enzymatically illuminating thegenerated pyrophosphate, and measuring the intensity of theillumination, and

(f) comparing the fluorescence intensity measured in step (e) with acontrol;

[10] the method of [1] or [2], comprising the following steps (a) to(f):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) synthesizing a probe complementary to a nucleotide sequencecomprising a sequence covering up to a nucleotide adjacent to a positionof any of (1) to (28) of [1], or a nucleotide in the complementarystrand composing a base pair with the nucleotide at the position,

(d) performing a single nucleotide extension reaction in the presence offluorescently labeled nucleotides, using the DNA amplified in step (b)as a template, and the primer synthesized in step (c),

(e) measuring the fluorescence polarization, and

(f) comparing the fluorescence polarization measured in step (e) with acontrol;

[11] the method of [1] or [2], comprising the following steps (a) to(f):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) synthesizing a primer complementary to a nucleotide sequencecomprising a sequence covering up to the nucleotide adjacent to aposition of any of (1) to (28) of [1], or a nucleotide in thecomplementary strand composing a base pair with the nucleotide at theposition,

(d) performing a single nucleotide extension reaction in the presence offluorescently labeled nucleotides, using the DNA amplified in step (b)as a template, and the primer synthesized in step (c),

(e) determining the nucleotide variety used in the reaction of step (d)using a sequencer, and

(f) comparing the nucleotide determined in step (e) with a control;

[12] the method of [1] or [2], comprising the following steps (a) to(d):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) measuring the molecular weight of the DNA amplified in step (b)using a mass spectrometer, and

(d) comparing the molecular weight measured in step (c) with a control;

[13] the method of [1] or [2], comprising the following steps (a) to(f):

(a) preparing DNA from a test rice,

(b) amplifying a DNA comprising a nucleotide in a position of any of (1)to (28) of [1], or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position,

(c) providing a substratum on which a nucleotide probe is immobilized,

(d) contacting the DNA of step (b) with the substratum of step (c),

(e) detecting the strength of hybridization between the DNA and thenucleotide probe immobilized on the substratum, and

(f) comparing the strength detected in step (e) with a control;

[14] the method of any of [1] to [13], further comprising the followingsteps (a) and (b):

(a) disrupting a rice seed in an alkaline aqueous solvent, and

(b) extracting rice genomic DNA from the seed disrupted in step (a);

[15] the method of [14], wherein the rice seed is polished;

[16] a primer for distinguishing between rice varieties (or a reagentfor distinguishing between rice varieties), wherein the primer is (a) anoligonucleotide for amplification of a DNA region comprising anucleotide in a position of any of (1) to (28) of [1] in the ricegenome, or a nucleotide in the complementary strand composing a basepair with the nucleotide at the position, or (b) an oligonucleotidecomprising a nucleotide sequence complementary to a sequence covering upto a nucleotide adjacent to a position of any of (1) to (28) of [1] inthe rice genome, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position;

[17] an oligonucleotide for distinguishing between rice varieties (or areagent for distinguishing between rice varieties), wherein theoligonucleotide hybridizes with a DNA region comprising a nucleotide ina position of any of (1) to (28) of [1], or a nucleotide in thecomplementary strand composing a base pair with the nucleotide at theposition, comprising at least 15 nucleotides;

[18] a kit for distinguishing between rice varieties, comprising theoligonucleotide of [16] or [17]; and

[19] the kit of [18], further comprising an alkaline aqueous solvent.

The inventors analyzed the genomic sequences of 24 rice varieties, andthus discovered polymorphic markers that enable the rice varieties to beaccurately distinguished. SEQ ID NOs: 1 to 28 show the DNA regions inthe rice genome that comprise the polymorphic sites identified by theinventors. The positions of each of the polymorphisms are shown in FIGS.1 to 29, and in Tables 8 and 9.

The present invention provides methods for distinguishing ricevarieties. In the methods, the type of a nucleotide at a polymorphicsite, identified by the inventors in the genome of 24 rice varieties, isfirst determined. More specifically, a nucleotide at any of thefollowing positions (1) to (28), or a nucleotide composing a base pairwith the above nucleotide at a position in the complementary strand, isdetermined in the rice genome (step (A)).

(1) Position 593 in the nucleotide sequence of SEQ ID NO: 1,

(2) Position 304 in the nucleotide sequence of SEQ ID NO: 2,

(3) Position 450 in the nucleotide sequence of SEQ ID NO: 3,

(4) Position 377 in the nucleotide sequence of SEQ ID NO: 4,

(5) Position 163 in the nucleotide sequence of SEQ ID NO: 5,

(6) Position 624 in the nucleotide sequence of SEQ ID NO: 6,

(7) Position 534 in the nucleotide sequence of SEQ ID NO: 7,

(8) Position 358 in the nucleotide sequence of SEQ ID NO: 8,

(9) Position 475 in the nucleotide sequence of SEQ ID NO: 9,

(10) Position 323 in the nucleotide sequence of SEQ ID NO: 10,

(11) Position 612 in the nucleotide sequence of SEQ ID NO: 11,

(12) Position 765 in the nucleotide sequence of SEQ ID NO: 12,

(13) Position 571 in the nucleotide sequence of SEQ ID NO: 13,

(14) Position 660 in the nucleotide sequence of SEQ ID NO: 14,

(15) Position 223 in the nucleotide sequence of SEQ ID NO: 15,

(16) Position 247 in the nucleotide sequence of SEQ ID NO: 16,

(17) Position 163 in the nucleotide sequence of SEQ ID NO: 17,

(18) Position 421 in the nucleotide sequence of SEQ ID NO: 18,

(19) Position 178 in the nucleotide sequence of SEQ ID NO: 19,

(20) Position 141 in the nucleotide sequence of SEQ ID NO: 20,

(21) Position 480 in the nucleotide sequence of SEQ ID NO: 21,

(22) Position 481 in the nucleotide sequence of SEQ ID NO: 22,

(23) Position 131 in the nucleotide sequence of SEQ ID NO: 23,

(24) Position 510 in the nucleotide sequence of SEQ ID NO: 24,

(25) Position 248 in the nucleotide sequence of SEQ ID NO: 25,

(26) Position 92 in the nucleotide sequence of SEQ ID NO: 26,

(27) Position 743 in the nucleotide sequence of SEQ ID NO: 27,

(28) Position 552 in the nucleotide sequence of SEQ ID NO: 28.

With information such as the nucleotide sequences and polymorphic sitesdisclosed herein, it is normally easy for one skilled in the art toappropriately identify an actual position on the genome that correspondsto a polymorphic site. For example, the genomic position of apolymorphism of this invention can be identified by referring topublicly available genome databases and the like. That is, even if aslight difference is found between a nucleotide sequence in the SequenceListing and an actual genomic sequence, the polymorphic sites of thisinvention on the actual genome can be precisely identified by performinghomology searches and such over the genomic sequence, using a nucleotidesequence shown in the Sequence Listing.

Normally, genomic DNA is composed of complementary double-stranded DNA.Therefore, herein, even when the DNA sequence of one strand is disclosedfor descriptive purposes, it should be naturally assumed that itscomplementary sequence (nucleotide) is also disclosed. When the DNAsequence (nucleotide) of one strand is known, its complementary sequence(nucleotide) is obvious to those skilled in the art.

Herein, a “polymorphism” is not limited to single nucleotidepolymorphisms (SNPs), comprising a mutation such as the substitution,deletion, and insertion of a single nucleotide. “Polymorphism” alsoincludes those of several continuous nucleotides. A “polymorphic marker”is herein defined as information on a nucleotide mutation at apolymorphic site (polymorphic mutation). More specifically, apolymorphic marker of this invention refers to information on a mutationin a nucleotide sequence, identified by comparing the genomic sequenceof rice variety Nipponbare with that of another variety, that can beused to distinguish the rice varieties. Herein, the polymorphic markersused to determine the type of a nucleotide are preferably thepolymorphic markers described in the following (1′) to (28′). Thus, in apreferred embodiment of this invention, rice varieties are distinguishedby using the polymorphic markers described in the following (1′) to(28′):

(1′) The nucleotide at position 593 in the nucleotide sequence of SEQ IDNO: 1 is T. More specifically, the nucleotide at position 593 in thenucleotide sequence of SEQ ID NO: 1 in the Nipponbare genome comprises aC to T substitution.

(2′) The nucleotide at position 304 in the nucleotide sequence of SEQ IDNO: 2 is T. More specifically, the nucleotide at position 304 in thenucleotide sequence of SEQ ID NO: 2 in the Nipponbare genome comprisesan A to T substitution.

(3′) The nucleotide at position 450 in the nucleotide sequence of SEQ IDNO: 3 is A. More specifically, the nucleotide at position 450 in thenucleotide sequence of SEQ ID NO: 3 in the Nipponbare genome comprises aG to A substitution.

(4′) The nucleotide at position 377 in the nucleotide sequence of SEQ IDNO: 4is C. More specifically, the nucleotide at position 377 in thenucleotide sequence of SEQ ID NO: 4 in the Nipponbare genome comprises aT to C substitution.

(5′) The nucleotide at position 163 in the nucleotide sequence of SEQ IDNO: 5 is C. More specifically, the nucleotide at position 163 in thenucleotide sequence of SEQ ID NO: 5 in the Nipponbare genome comprises aT to C substitution.

(6′) The nucleotide at position 624 in the nucleotide sequence of SEQ IDNO: 6 is C. More specifically, the nucleotides at positions 624 to 626in the nucleotide sequence of SEQ ID NO: 6 in the Nipponbare genome aredeleted.

(7′) The nucleotide at position 534 in the nucleotide sequence of SEQ IDNO: 7 is C. More specifically, the nucleotide at position 534 in thenucleotide sequence of SEQ ID NO: 7 in the Nipponbare genome comprisesan A to C substitution.

(8′) The nucleotide at position 358 in the nucleotide sequence of SEQ IDNO: 8 is G. More specifically, GT is inserted between the nucleotides atpositions 358 and 389 in the nucleotide sequence of SEQ ID NO: 8 in theNipponbare genome.

(9′) The nucleotide at position 475 in the nucleotide sequence of SEQ IDNO: 9 is G. More specifically, the nucleotide at position 475 in thenucleotide sequence of SEQ ID NO: 9 in the Nipponbare genome comprises aT to G substitution.

(10′) The nucleotide at position 323 in the nucleotide sequence of SEQID NO: 10 is A. More specifically, the nucleotide at position 323 in thenucleotide sequence of SEQ ID NO: 10 in the Nipponbare genome comprisesa G to A substitution.

(11′) The nucleotide at position 612 in the nucleotide sequence of SEQID NO: 11 is A. More specifically, the nucleotides at positions 612 and613 in the nucleotide sequence of SEQ ID NO: 11 in the Nipponbare genomeare substituted from CA to AG.

(12′) The nucleotide at position 765 in the nucleotide sequence of SEQID NO: 12 is T. More specifically, the nucleotide at position 765 in thenucleotide sequence of SEQ ID NO: 12 in the Nipponbare genome comprisesa G to T substitution.

(13′) The nucleotide at position 571 in the nucleotide sequence of SEQID NO: 13 is T. More specifically, the nucleotide at position 571 in thenucleotide sequence of SEQ ID NO: 13 in the Nipponbare genome comprisesa G to T substitution.

(14′) The nucleotide at position 660 in the nucleotide sequence of SEQID NO: 14 is G. More specifically, the nucleotide at position 660 in thenucleotide sequence of SEQ ID NO: 14 in the Nipponbare genome comprisesan A to G substitution.

(15′) The nucleotide at position 223 in the nucleotide sequence of SEQID NO: 15 is A. More specifically, the nucleotide at position 223 in thenucleotide sequence of SEQ ID NO: 15 in the Nipponbare genome comprisesa G to A substitution.

(16′) The nucleotide at position 247 in the nucleotide sequence of SEQID NO: 16 is A. More specifically, the nucleotide at position 247 in thenucleotide sequence of SEQ ID NO: 16 in the Nipponbare genome comprisesa G to A substitution.

(17′) The nucleotide at position 163 in the nucleotide sequence of SEQID NO: 17 is A. More specifically, the nucleotide at position 163 in thenucleotide sequence of SEQ ID NO: 17 in the Nipponbare genome comprisesa G to A substitution.

(18′) The nucleotide at position 421 in the nucleotide sequence of SEQID NO: 18 is C. More specifically, the nucleotide at position 421 in thenucleotide sequence of SEQ ID NO: 18 in the Nipponbare genome comprisesan A to C substitution.

(19′) The nucleotide at position 178 in the nucleotide sequence of SEQID NO: 19 is G. More specifically, the nucleotide at position 178 in thenucleotide sequence of SEQ ID NO: 19 in the Nipponbare genome isdeleted.

(20′) The nucleotide at position 141 in the nucleotide sequence of SEQID NO: 20 is G. More specifically, the nucleotide at position 141 in thenucleotide sequence of SEQ ID NO: 20 in the Nipponbare genome comprisesan A to G substitution.

(21′) The nucleotide at position 480 in the nucleotide sequence of SEQID NO: 21 is C. More specifically, the nucleotide at position 480 in thenucleotide sequence of SEQ ID NO: 21 in the Nipponbare genome comprisesa T to C substitution.

(22′) The nucleotide at position 481 in the nucleotide sequence of SEQID NO: 22 is C. More specifically, the nucleotide at position 481 in thenucleotide sequence of SEQ ID NO: 22 in the Nipponbare genome comprisesa T to C substitution.

(23′) The nucleotide at position 131 in the nucleotide sequence of SEQID NO: 23 is C. More specifically, the nucleotide at position 131 in thenucleotide sequence of SEQ ID NO: 23 in the Nipponbare genome comprisesa G to C substitution.

(24′) The nucleotide at position 510 in the nucleotide sequence of SEQID NO: 24 is A. More specifically, the nucleotide at position 510 in thenucleotide sequence of SEQ ID NO: 24 in the Nipponbare genome comprisesa G to A substitution.

(25′) The nucleotide at position 248 in the nucleotide sequence of SEQID NO: 25 is T. More specifically, the nucleotide at position 248 in thenucleotide sequence of SEQ ID NO: 25 in the Nipponbare genome comprisesa C to T substitution.

(26′) The nucleotide at position 92 in the nucleotide sequence of SEQ IDNO: 26 is C. More specifically, the nucleotide at position 92 in thenucleotide sequence of SEQ ID NO: 26 in the Nipponbare genome comprisesa G to C substitution.

(27′) The nucleotide at position 743 in the nucleotide sequence of SEQID NO: 27 is G. More specifically, the nucleotide at position 743 in thenucleotide sequence of SEQ ID NO: 27 in the Nipponbare genome comprisesan A to G substitution.

(28′) The nucleotide at position 552 in the nucleotide sequence of SEQID NO: 28 is T. More specifically, the nucleotide at position 552 in thenucleotide sequence of SEQ ID NO: 28 in the Nipponbare genome comprisesa C to T substitution.

Herein, “determining the type of nucleotide” normally means determiningthe nucleotide sequence at a position described in any of the above (1)to (28) on the genome of a rice whose variety is being distinguished(also described below as a “test rice”). However, it may not benecessary to specifically determine the actual species of thenucleotide. Even when a nucleotide sequence at any of the abovepositions (1) to (28) is not specifically determined in the genome of atest rice, it is possible to distinguish the rice variety by examiningwhether or not it is identical to that of Nipponbare.

Next, in the methods of this invention, a nucleotide sequence determinedin the above step (A) is related to the rice varieties (step (B)).

The rice varieties that can be distinguished by the methods of thisinvention are as follows (the name of each variety may be abbreviatedherein, as shown in the parentheses) : Nipponbare (nhb), Hatsushimo(hts), Mutsuhomare (mth), Yukinosei (yki), Kirara 397 (krr),Tsugaruroman (tgr), Gohyakumangoku (ghm), Morinokumasan (mnk), Yumeakari(yma), Hanaechizen (hez), Koshihikari (ksh), Tsukinohikari (tkh),Akitakomachi (akk), Asanohikari (ash), Aichinokaori (ank), Matsuribare(mtb), Hinohikari (hnh), Yumetsukushi (ymt), Hitomebore (hit),Manamusume (mmm), Fusaotome (fom), Dontokoi (don), Kinuhikari (knh),Sasanishiki (ssk), Akebono (akb), and Goropikari (grp).

The methods of this invention for distinguishing rice varieties may benormally used to identify the name of a rice of unknown variety,selected from the above varieties, or to determine whether a ricebelongs to one of the above varieties.

The present inventors determined the nucleotide sequences at positionsdescribed in the above (1) to (28) in the genome of the above ricevarieties, and obtained polymorphic markers. Table 1 shows the detailsof the polymorphic markers (the names of the polymorphic markers, andthe nucleotide sequences at the above positions (1) to (28) for each ofthe rice varieties). TABLE 1 SEQ SNP Detection Marker ID NipponbareHatsushimo Mutsuhomare Yukinosei Kirara 397 Tsugaruroman GohyakumangokuMorinokumasan name NO: Position nhb hts mth yki krr tgr ghm mnk S0015 1593 C C C C T T C T S0040 2 304 T T T A A A A A S0279 3 450 C T T T T TT T S0044 4 377 T T T T T T T T S0252 5 163 T C C C C T T T S0109 6 624T T C T C C C C S0115 7 534 T G G G G T T G S0107 8 358 A A G G G G G GS0126 9 475 T T T G T T T T S0124 10 323 G G G G A G G G S0146 11 612 CC C C A A C C S0135 12 765 G G G G G G T G S0155 13 571 G G T G T T T TS0161 14 660 A G G A A G A A S0177 15 223 G A A A A A A A S0178 16 247 CC C C T C T C S0174 17 163 G G G G G A G A S0185 18 421 A A A A A C C CS0208 19 178 C G G G G G G G S0007 20 141 A A A G G A G A S0070 21 480 AA G A A A G A S0310 22 481 T T T T C C C C S0375 23 131 G G C G G C G CS0346 24 510 G G G G A G A G S0013 25 248 C C C C T C C C S0347 26 92 CC G C G C G C S0330 27 743 A A A A G A A A S0336 28 552 C C T C T T C TSNP Detection Marker Yumeakari Hanaechizen Koshihikari TsukinohikariAkitakomachi Asanohikari Aichinokaori Matsuribare Hinohikari name ymahez ksh tkh akk ash ank mtb hnh S0015 T C T T T C C T C S0040 A A A T AT T T A S0279 T T T C T T T T T S0044 T T C T T T T T T S0252 C C T T TT C T T S0109 C T C T C T C T C S0115 T G G T G T G T G S0107 G G G G GG G G G S0126 G T T T T T T T T S0124 G G G G G G G G G S0146 A C A A AA C A C S0135 G G T G G G G G G S0155 T G G T T G G T T S0161 G A A A GA G A A S0177 A G A G A A A A A S0178 C C T T C C C C C S0174 A A A G AG G G A S0185 C C C C C C C C C S0208 G C G C G C G G G S0007 A A A A AA A A A S0070 A G A A A A A A A S0310 C T C T C T T T T S0375 G G G C GC G C C S0346 G G A A G G G G G S0013 C C C C C C C C C S0347 G G G G GC G G C S0330 A A A A A A A A A S0336 T T T C T C C C T SNP DetectionMarker Yumetsukushi Hitomebore Manamusume Fusaotome Dontokoi KinuhikariSasanishiki Akebono Goropikari name ymt hit mmm fom don knh ssk akb grpS0015 C T T T T C T C T S0040 T A A A T T A T A S0279 T T T T T T T T TS0044 C T T T C C T T T S0252 T T C C C T T C T S0109 C C C C C C T T TS0115 G G G G T G T T G S0107 G G G G G G G A G S0126 T T T T T T G T TS0124 G G G G G G G G G S0146 A C C C A A C C C S0135 T T T T T T G G GS0155 G G G G T G G G T S0161 A A A A A A A G A S0177 A A A A A A A A GS0178 T T T T T T C C T S0174 A A A A A A G G G S0185 A C C C A A C C CS0208 G G G C G G G G C S0007 A A A A A A A G A S0070 A A A A G A A G AS0310 C C C C C C T T C S0375 G C C G C G C C C S0346 A A A A A A G G AS0013 C C C C C C C T C S0347 G G G G G G G C G S0330 A A A A A A A A AS0336 C T T T C C T C T

Herein, the variety of a test rice can be identified by determining thenucleotide sequence at an above position (1) to (28) in its genome, andcomparing that with data on the nucleotide sequences of the ricevarieties, shown in Table 1. In a preferred embodiment of thisinvention, a nucleotide sequence is distinguished by using a polymorphicmarker described in the above (1′) to (28′). In the methods of thisinvention, it is not necessary to determine the nucleotide sequences atall positions described in (1) to (28) above. For example, thepolymorphic marker “S0124” may be used to determine the nucleotidesequence at position 323 in the nucleotide sequence of SEQ ID NO: 10, asshown in (10), above. If the nucleotide sequence is determined to be A(adenine), the test rice is identified as Kirara 397. In another case,the polymorphic markers “S0126” and “S0015” may be used in combinationto determine the nucleotide sequences. If G is the nucleotide atposition 475 in the nucleotide sequence of SEQ ID NO: 9, in (9) above;and C is the nucleotide at position 593 in the nucleotide sequence ofSEQ ID NO: 1, in (1) above, then the test rice is identified asYukinosei. Thus, using the nucleotide sequences determined in the genomeof a test rice at positions described in the above (1) to (28), oneskilled in the art can easily determine the rice variety based on Table1, provided herein.

Furthermore, in the methods of this invention, it is not necessary todetermine the type of nucleotide at the positions described in (1) to(28), above. Rice varieties may be distinguished by examining whether anucleotide sequence, at an above position (1) to (28) in the genome of atest rice, is identical to a Nipponbare sequence at the same position.In a preferred embodiment of this invention, the polymorphic markers ofthe above (1′) to (28′) may be used to determine whether the nucleotidesequence in a test rice genome at a position (1) to (28) above, isidentical to a Nipponbare sequence at this position.

For each of the above rice varieties, the present inventors examinedwhether or not the nucleotide sequences at positions (1) to (28) aboveare identical to those of Nipponbare, and they established combinationsof polymorphic markers that enable the above varieties to bedistinguished (Tables 2 to 7). In Tables 2 to 7, the combinations ofpolymorphic markers that can distinguish varieties are shaded.Combinations of polymorphic markers are not limited to those shown inTables 2 to 7, and those skilled in the art can appropriately selectcombinations of polymorphic markers that can be used to distinguishvarieties, according to nucleotide sequence information at positions (1)to (28) above in the genome of 26 rice varieties, providedbythepresentinvention. In the tables, a circle shows a match with Nipponbare, whilea cross indicates a mismatch. Table 2 Marker Nipponbare HatsushimoMutsuhomare Yukinosei Kirara 397 Tsugaruroman GohyakumangokuMorinokumasan Yumeakari Nipponbare S0107

◯ X X X X X X X S0177

X X X X X X X X Hatsushimo S0107 ◯

X X X X X X X S0177 ◯

X X X X X X X S0185 ◯

◯ ◯ ◯ X X X X Mutsuhomare S0070 ◯ ◯

◯ ◯ ◯ X ◯ ◯ S0161 ◯ X

◯ ◯ X ◯ ◯ X S0109 ◯ ◯

◯ X X X X X Yukinosei S0126 ◯ ◯ ◯

◯ ◯ ◯ ◯ X S0015 ◯ ◯ ◯

X X ◯ X X Kirara 397 S0124 ◯ ◯ ◯ ◯

◯ ◯ ◯ ◯ Marker Hanaechizen Koshihikari Tsukinohikari AkitakomachiAsanohikari Aichinokaori Matsuribare Hinohikari Yumetsukushi NipponbareS0107 X X X X X X X X X S0177 ◯ X ◯ X X X X X X Hatsushimo S0107 X X X XX X X X X S0177 ◯ X ◯ X X X X X X S0185 X X X X X X X X ◯ MutsuhomareS0070 X ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ S0161 ◯ ◯ ◯ X ◯ X ◯ ◯ ◯ S0109 ◯ X ◯ X ◯ X ◯ X XYukinosei S0126 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ S0015 ◯ X X X ◯ ◯ X ◯ ◯ Kirara 397S0124 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Marker Hitomebore Manamusume Fusaotome DontokoiKinuhikari Sasanishiki Akebono Goropikari Nipponbare S0107 X X X X X X ◯X S0177 X X X X X X X ◯ Hatsushimo S0107 X X X X X X ◯ X S0177 X X X X XX X ◯ S0185 X X X ◯ ◯ X X X Mutsuhomare S0070 ◯ ◯ ◯ X ◯ ◯ X ◯ S0161 ◯ ◯◯ ◯ ◯ ◯ X ◯ S0109 X X X X X ◯ ◯ ◯ Yukinosei S0126 ◯ ◯ ◯ ◯ ◯ X ◯ ◯ S0015X X X X ◯ X ◯ X Kirara 397 S0124 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯

TABLE 3 Marker Nipponbare Hatsushimo Mutsuhomare Yukinosei Kirara 397Tsugaruroman Gohyakumangoku Morinokumasan Yumeakari Tsugaruroman S0161 ◯X X ◯ ◯

◯ ◯ X S0252 ◯ X X X X

◯ ◯ X S0115 ◯ X X X X

◯ X ◯ Gohyakumangoku S0135 ◯ ◯ ◯ ◯ ◯ ◯

◯ ◯ S0174 ◯ ◯ ◯ ◯ ◯ X

X X Morinokumasan S0178 ◯ ◯ ◯ ◯ X ◯ X

◯ S0015 ◯ ◯ ◯ ◯ X X ◯

X S0161 ◯ X X ◯ ◯ X ◯

X S0109 ◯ ◯ X ◯ X X X

X Yumeakari S0126 ◯ ◯ ◯ X ◯ ◯ ◯ ◯

S0109 ◯ ◯ X ◯ X X X X

Hanaechizen S0177 ◯ X X X X X X X X S0174 ◯ ◯ ◯ ◯ ◯ X ◯ X X MarkerHanaechizen Koshihikari Tsukinohikari Akitakomachi AsanohikariAichinokaori Matsuribare Hinohikari Yumetsukushi Tsugaruroman S0161 ◯ ◯◯ X ◯ X ◯ ◯ ◯ S0252 X ◯ ◯ ◯ ◯ X ◯ ◯ ◯ S0115 X X ◯ X ◯ X ◯ X XGohyakumangoku S0135 ◯ X ◯ ◯ ◯ ◯ ◯ ◯ X S0174 X X ◯ X ◯ ◯ ◯ X XMorinokumasan S0178 ◯ X X ◯ ◯ ◯ ◯ ◯ X S0015 ◯ X X X ◯ ◯ X ◯ ◯ S0161 ◯ ◯◯ X ◯ X ◯ ◯ ◯ S0109 ◯ X ◯ X ◯ X ◯ X X Yumeakari S0126 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯S0109 ◯ X ◯ X ◯ X ◯ X X Hanaechizen S0177

X ◯ X X X X X X S0174

X ◯ X ◯ ◯ ◯ X X Marker Hitomebore Manamusume Fusaotome DontokoiKinuhikari Sasanishiki Akebono Goropikari Tsugaruroman S0161 ◯ ◯ ◯ ◯ ◯ ◯X ◯ S0252 ◯ X X X ◯ ◯ X ◯ S0115 X X X ◯ X ◯ ◯ X Gohyakumangoku S0135 X XX X X ◯ ◯ ◯ S0174 X X X X X ◯ ◯ ◯ Morinokumasan S0178 X X X X X ◯ ◯ XS0015 X X X X ◯ X ◯ X S0161 ◯ ◯ ◯ ◯ ◯ ◯ X ◯ S0109 X X X X X ◯ ◯ ◯Yumeakari S0126 ◯ ◯ ◯ ◯ ◯ X ◯ ◯ S0109 X X X X X ◯ ◯ ◯ Hanaechizen S0177X X X X X X X ◯ S0174 X X X X X ◯ ◯ ◯

TABLE 4 Marker Nipponbare Hatsushimo Mutsuhomare Yukinosei Kirara 397Tsugaruroman Gohyakumangoku Morinokumasan Yumeakari Koshihikari S0040 ◯◯ ◯ X X X X X X S0044 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Tsukinohikari S0279 ◯ X X X X XX X X S0107 ◯ ◯ X X X X X X X Akitakomachi S0115 ◯ X X X X ◯ ◯ X ◯ S0146◯ ◯ ◯ ◯ X X ◯ ◯ X S0178 ◯ ◯ ◯ ◯ X ◯ X ◯ ◯ Asanohikari S0208 ◯ X X X X XX X X S0146 ◯ ◯ ◯ ◯ X X ◯ ◯ X S0177 ◯ X X X X X X X X Marker HanaechizenKoshihikari Tsukinohikari Akitakomachi Asanohikari AichinokaoriMatsuribare Hinohikari Yumetsukushi Koshihikari S0040 X

◯ X ◯ ◯ ◯ X ◯ S0044 ◯

◯ ◯ ◯ ◯ ◯ ◯ X Tsukinohikari S0279 X X

X X X X X X S0107 X X

X X X X X X Akitakomachi S0115 X X ◯

◯ X ◯ X X S0146 ◯ X X

X ◯ X ◯ X S0178 ◯ X X

◯ ◯ ◯ ◯ X Asanohikari S0208 ◯ X ◯ X

X X X X S0146 ◯ X X X

◯ X ◯ X S0177 ◯ X ◯ X

X X X X Marker Hitomebore Manamusume Fusaotome Dontokoi KinuhikariSasanishiki Akebono Goropikari Koshihikari S0040 X X X ◯ ◯ X ◯ X S0044 ◯◯ ◯ X X ◯ ◯ ◯ Tsukinohikari S0279 X X X X X X X X S0107 X X X X X X ◯ XAkitakomachi S0115 X X X ◯ X ◯ ◯ X S0146 ◯ ◯ ◯ X X ◯ ◯ ◯ S0178 X X X X X◯ ◯ X Asanohikari S0208 X X ◯ X X X X ◯ S0146 ◯ ◯ ◯ X X ◯ ◯ ◯ S0177 X XX X X X X ◯

TABLE 5 Marker Nipponbare Hatsushimo Mutsuhomare Yukinosei Kirara 397Tsugaruroman Gohyakumangoku Morinokumasan Yumeakari Aichinokaori S0109 ◯◯ X ◯ X X X X X S0155 ◯ ◯ X ◯ X X X X X S0104 ◯ ◯ ◯ ◯ ◯ X ◯ X XMatsuribare S0109 ◯ ◯ X ◯ X X X X X S0208 ◯ X X X X X X X X S0146 ◯ ◯ ◯◯ X X ◯ ◯ X Hinohikari S0015 ◯ ◯ ◯ ◯ X X ◯ X X S0155 ◯ ◯ X ◯ X X X X XS0174 ◯ ◯ ◯ ◯ ◯ X ◯ X X Marker Hanaechizen Koshihikari TsukinohikariAkitakomachi Asanohikari Aichinokaori Matsuribare HinohikariYumetsukushi Aichinokaori S0109 ◯ X ◯ X ◯

◯ X X S0155 ◯ ◯ X X ◯

X X ◯ S0104 X X ◯ X ◯

◯ X X Matsuribare S0109 ◯ X ◯ X ◯ X

X X S0208 ◯ X ◯ X ◯ X

X X S0146 ◯ X X X X ◯

◯ X Hinohikari S0015 ◯ X X X ◯ ◯ X

◯ S0155 ◯ ◯ X X ◯ ◯ X

◯ S0174 X X ◯ X ◯ ◯ ◯

X Marker Hitomebore Manamusume Fusaotome Dontokoi Kinuhikari SasanishikiAkebono Goropikari Aichinokaori S0109 X X X X X ◯ ◯ ◯ S0155 ◯ ◯ ◯ X ◯ ◯◯ X S0104 X X X X X ◯ ◯ ◯ Matsuribare S0109 X X X X X ◯ ◯ ◯ S0208 ◯ ◯ ◯X ◯ ◯ ◯ X S0146 ◯ ◯ ◯ X X ◯ ◯ ◯ Hinohikari S0015 X X X X ◯ X ◯ X S0155 ◯◯ ◯ X ◯ ◯ ◯ X S0174 X X X X X ◯ ◯ ◯

TABLE 6 Marker Nipponbare Hatsushimo Mutsuhomare Yukinosei Kirara 397Tsugaruroman Gohyakumangoku Morinokumasan Yumeakari Hitomebore S0044 ◯ ◯◯ ◯ ◯ ◯ ◯ ◯ ◯ S0135 ◯ ◯ ◯ ◯ ◯ ◯ X ◯ ◯ S0115 ◯ X X X X ◯ ◯ X ◯ S0252 ◯ XX X X ◯ ◯ ◯ X Manamusume S0135 ◯ ◯ ◯ ◯ ◯ ◯ X ◯ ◯ S0208 ◯ X X X X X X X XS0252 ◯ X X X X ◯ ◯ ◯ X S0146 ◯ ◯ ◯ ◯ X X ◯ ◯ X Fusaotome S0135 ◯ ◯ ◯ ◯◯ ◯ X ◯ ◯ S0208 ◯ X X X X X X X X Dontokoi S0044 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ S0252◯ X X X X ◯ ◯ ◯ X Marker Hanaechizen Koshihikari TsukinohikariAkitakomachi Asanohikari Aichinokaori Matsuribare HinohikariYumetsukushi Hitomebore S0044 ◯ X ◯ ◯ ◯ ◯ ◯ ◯ X S0135 ◯ X ◯ ◯ ◯ ◯ ◯ ◯ XS0115 X X ◯ X ◯ X ◯ X X S0252 X ◯ ◯ ◯ ◯ X ◯ ◯ ◯ Manamusume S0135 ◯ X ◯ ◯◯ ◯ ◯ ◯ X S0208 ◯ X ◯ X ◯ X X X X S0252 X ◯ ◯ ◯ ◯ X ◯ ◯ ◯ S0146 ◯ X X XX ◯ X ◯ X Fusaotome S0135 ◯ X ◯ ◯ ◯ ◯ ◯ ◯ X S0208 ◯ X ◯ X ◯ X X X XDontokoi S0044 ◯ X ◯ ◯ ◯ ◯ ◯ ◯ X S0252 X ◯ ◯ ◯ ◯ X ◯ ◯ ◯ MarkerHitomebore Manamusume Fusaotome Dontokoi Kinuhikari Sasanishiki AkebonoGoropikari Hitomebore S0044

◯ ◯ X X ◯ ◯ ◯ S0135

X X X X ◯ ◯ ◯ S0115

X X ◯ X ◯ ◯ X S0252

X X X ◯ ◯ X ◯ Manamusume S0135 X

X X X ◯ ◯ ◯ S0208 X

◯ X X X X ◯ S0252 ◯

X X ◯ ◯ X ◯ S0146 ◯

◯ X X ◯ ◯ ◯ Fusaotome S0135 X X

X X ◯ ◯ ◯ S0208 X X

X X X X ◯ Dontokoi S0044 ◯ ◯ ◯

X ◯ ◯ ◯ S0252 ◯ X X

◯ ◯ X ◯

TABLE 7 Marker Nipponbare Hatsushimo Mutsuhomare Yukinosei Kirara 397Tsugaruroman Gohyakumangoku Morinokumasan Yumeakari Yumetsukushi -Kinuhikari S0044 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ S0015 ◯ ◯ ◯ ◯ X X ◯ X X SasanishikiS0126 ◯ ◯ ◯ X ◯ ◯ ◯ ◯ X S0252 ◯ X X X X ◯ ◯ ◯ X Akebono S0161 ◯ X X ◯ ◯X ◯ ◯ X S0007 ◯ ◯ ◯ X X ◯ X ◯ ◯ Goropikari S0177 ◯ X X X X X X X X S0155◯ ◯ X ◯ X X X X X S0115 ◯ X X X X ◯ ◯ X ◯ Marker Hanaechizen KoshihikariTsukinohikari Akitakomachi Asanohikari Aichinokaori MatsuribareHinohikari Yumetsukushi Yumetsukushi - Kinuhikari S0044 ◯ X ◯ ◯ ◯ ◯ ◯ ◯

S0015 ◯ X X X ◯ ◯ X ◯

Sasanishiki S0126 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ S0252 X ◯ ◯ ◯ ◯ X ◯ ◯ ◯ AkebonoS0161 ◯ ◯ ◯ X ◯ X ◯ ◯ ◯ S0007 ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ ◯ Goropikari S0177 ◯ X ◯ XX X X X X S0155 ◯ ◯ X X ◯ ◯ X X ◯ S0115 X X ◯ X ◯ X ◯ X X MarkerHitomebore Manamusume Fusaotome Dontokoi Kinuhikari Sasanishiki AkebonoGoropikari Yumetsukushi - Kinuhikari S0044 ◯ ◯ ◯ X

◯ ◯ ◯ S0015 X X X X

X ◯ X Sasanishiki S0126 ◯ ◯ ◯ ◯ ◯

◯ ◯ S0252 ◯ X X X ◯

X ◯ Akebono S0161 ◯ ◯ ◯ ◯ ◯ ◯

◯ S0007 ◯ ◯ ◯ ◯ ◯ ◯

◯ Goropikari S0177 X X X X X X X

S0155 ◯ ◯ ◯ X ◯ ◯ ◯

S0115 X X X ◯ X ◯ ◯

For example, a test rice may be examined using the polymorphic marker“S0135” for the nucleotide sequence at position 765 in the nucleotidesequence of SEQ ID NO: 12, as shown in the above (12), and “S0208” forthe nucleotide sequence at position 178 of SEQ ID NO: 19, as shown in(19). If the nucleotide sequence of a test rice does not match with thatof Nipponbare at the former site, but matches at the latter site, thenthe test rice is identified as Fusaotome. By determining the nucleotidesequences at the above positions using each of the polymorphic markers(1′) to (28′) above, the nucleotide sequence of a test rice andNipponbare at a position (1) to (28) above can be found to match, or notto match, and the variety of a test rice can be easily distinguishedbased on Tables 2 to 7.

One skilled in the art can determine the nucleotide species in the abovestep (A) of this invention using a publicly known method for determiningnucleotide sequences, a method for detecting polymorphic mutations, orthe like. For example, in a preferred embodiment of this invention, thefollowing method may be used: First, DNAs are prepared from a test rice.Herein, a test rice includes its leaf, root, seed, callus, leaf sheath,cultured cell, and the like, but it is not limited thereto. Furthermore,one skilled in the art may prepare DNAs from chromosomal DNAs extractedfrom the test rice. For example, in a preferred method, rice seeds maybe disrupted in an alkaline aqueous solution, and then genomic DNAs maybe extracted from the disrupted seeds; however, the methods are notlimited thereto. In the above, the seeds are preferably polished.

In these methods, a DNA comprising a nucleotide at a position describedin any of the above (1) to (28), or a nucleotide composing a base pairwith the above nucleotide at a position on the complementary strand, isthen amplified. Herein, a method for amplifying a DNA may be PCR, but isnot limited thereto, as long as it enables DNA amplification.

In this method, the nucleotide sequence of an amplified DNA is thendetermined. Nucleotide sequences can be determined by methods commonlyknown to those skilled in the art.

In these methods, a determined nucleotide sequence is then compared withthat of a control. Herein, the control is normally Nipponbare, which isrepresented by the sequences described in SEQ ID NO: 1 to NO: 28.Alternatively, one skilled in the art may obtain the nucleotide sequenceinformation of a wild type Nipponbare genome from a variety of genedatabases, references, or the like. In this method, polymorphisms aredetermined to be present or absent in a test rice genome by comparisonwith a control.

The methods for distinguishing rice varieties of this invention may beperformed by a variety of methods for enabling polymorphism detection,instead of by directly determining the nucleotide sequence of a DNAderived from a test rice, as described above. For example, the methodsof this invention for distinguishing rice varieties may be performedusing the following methods:

First, DNAs are prepared from a test rice. Then, the prepared DNA isdigested with a restriction enzyme. The DNA fragments are thenfractionated by size, and the sizes of the detected fragments arecompared with a control. In an alternative embodiment, DNAs are firstprepared from a test rice. Then, DNA comprising a nucleotide describedin any of the above (1) to (28), or a nucleotide composing a base pairwith the above nucleotide in the complementary strand, is amplified.Then, the amplified DNA is digested with a restriction enzyme. The DNAfragments are then fractionated by size, and the sizes of the detectedDNA fragments are compared with a control.

Examples of such methods are methods utilizing RFLP (restrictionfragment length polymorphism), PCR-RFLP, or the like. Specifically, if amutation exists in a restriction enzyme recognition site, or if a DNAfragment generated by restriction enzyme treatment comprises a baseinsertion or deletion, then the size of the fragment that results fromrestriction enzyme treatment should be different from that of a control.A region comprising such a mutation may be amplified by PCR, and treatedwith the corresponding restriction enzyme to detect the mutation bydifferences in band mobility after electrophoresis. Alternatively,chromosomal DNA may be treated with such restriction enzymes, separatedby electrophoresis, and then Southern blotting may be performed using anoligonucleotide of this invention to detect the presence or absence of amutation. One skilled in the art can appropriately choose restrictionenzymes according to the mutations.

Furthermore, in another method, DNAs are first prepared from a testrice. Then, a DNA comprising a nucleotide described in any of the above(1) to (28), or a nucleotide composing a base pair with the abovenucleotide on the complementary strand, is amplified. The amplified DNAis then dissociated into single strands. Dissociated single-strand DNAis separated on a non-denaturing gel. The mobility of the separatedsingle-strand DNA on the gel is compared with that of a control.

An example of the above method is PCR-SSCP (single-strand conformationpolymorphism) (Cloning and polymerase chain reaction-single-strandconformation polymorphism analysis of anonymous Alu repeats onchromosome 11. Genomics 12(1): 139-146 (1992, Jan. 1); Detection of p53gene mutations in human brain tumors by single-strand conformationpolymorphism analysis of polymerase chain reaction products. Oncogene6(8): 1313-1318 (1991, Aug. 1); Multiple fluorescence-based PCR-SSCPanalysis with postlabeling., PCR Methods Appl. 4(5): 275-282 (1995, Apr.1)). This method is particularly suitable for screening a large numberof DNA samples, due to advantages such as the relative ease ofmanipulation and the small amount of sample required. The principle ofthe method is as follows: When a double-stranded DNA fragment isdissociated into single strands, each strand gives rise to a uniqueconformation, according to its nucleotide sequence. Thus, when thedissociated DNA strands are separated by electrophoresis on anon-denaturing polyacrylamide gel, complementary single-stranded DNAs ofthe same length migrate to different positions, according to thedifferences in their respective conformations. The substitution of asingle nucleotide can change the conformation of a single-stranded DNA,resulting in a different mobility during electrophoresis on apolyacrylamide gel. Thus, the presence of a mutation in a DNA fragment,such as a point mutation, deletion, and insertion, can be detected bydetecting the mobility shift.

Specifically, a DNA comprising a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide on the complementary strand, is first amplified by PCR or thelike. Normally, a region for amplification is preferably about 200 bp to400 bp long. One skilled in the art can perform PCR by appropriatelyselecting reaction conditions and the like. An amplified DNA product maybe labeled during PCR using a primer labeled with an isotope such as³²P, or a fluorescent dye, biotin, or the like. Alternatively, anamplified DNA product may be labeled by performing PCR in whichnucleotide substrates labeled with an isotope such as ³²P, or afluorescent dye, biotin, or the like have been added to the PCR mixture.Furthermore, amplified DNA fragments may be labeled after PCR by usingKlenow enzyme or the like to attach nucleotide substrates labeled withan isotope such as ³²P, or a fluorescent dye, biotin, or the like. Theresulting labeled DNA fragment is denatured by heating, for example, andsubjected to electrophoresis on a polyacrylamide gel, without adenaturing agent such as urea. Conditions for separating DNA fragmentsmay be improved by adding an appropriate amount of glycerol (about 5 to10%) to the polyacrylamide gel. Conditions for electrophoresis maydiffer according to the properties of each DNA fragment; normallyelectrophoresis is performed at room temperature (20 to 25° C.). If adesired separation is not achieved, temperatures between 4 and 30° C.may be tested for optimal mobility. Following electrophoresis, themobilities of DNA fragments are detected and analyzed by autoradiographyusing X-ray film, or by scanning on a scanner for fluorescencedetection, or the like. When a band with different mobility is detected,it is directly cut from the gel, re-amplified using PCR, and subjectedto direct sequencing to confirm the presence of a mutation. In addition,if not using labeled DNAs, the bands may be detected by staining thegels after electrophoresis with ethidium bromide, by silver staining, orthe like.

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Next, two different probes, oligonucleotides complementaryto a nucleotide sequence near a DNA comprising a nucleotide sequencedescribed in any of the above (1) to (28), or near to a nucleotidecomposing a base pair with the above nucleotide in the complementarystrand, which are labeled with a fluorescent reporter and fluorescencequencher, are synthesized (step (b)). Then (step (c)), the DNA preparedin step (a) is hybridized with the probes synthesized in step (b). A DNAcomprising a nucleotide described in any of the above (1) to (28), or anucleotide composing a base pair with the above nucleotide in thecomplementary strand, is then amplified (step (d)), and reporterfluorescence emission is detected (step (e)). Then (step (f)), thefluorescent reporter emission detected in step (e) is compared with thatof a control.

An example of such methods is TaqMan PCR (Strategies in SNP genepolymorphism. Kennichi Matsubara and Yoshiyuki Sakaki. Nakayama-Shotenp94-105; Genet. Anal. 14: 143-149 (1999)). Specifically, the 5′-end ofprobe is first labeled with a fluorescent reporter. Herein, fluorescentreporters include FAM and VIC, but are not limited thereto. In addition,the 3′-end of the above probe is labeled with a fluorescence quencher.Herein, a fluorescence quenchers may be any substance that can quench afluorescent reporter. Then, probes labeled with the fluorescent reporterand fluorescence quencher are hybridized with the prepared DNA.Hybridization is normally performed under stringent conditions.Stringent conditions are, for example, normally 42° C., 2×SSC and 0.1%SDS, preferably 50° C., 2×SSC and 0.1% SDS, and more preferably 65° C.,0.1×SSC and 0.1% SDS, but are not limited thereto. A number of factorssuch as temperature and salt concentration can affect hybridizationstringency, and one skilled in the art can achieve optimal stringenciesby appropriately selecting the above factors.

A DNA comprising a nucleotide sequence described in any of the above (1)to (28), or a nucleotide composing a base pair with the above nucleotidein the complementary strand, is amplified using a DNA polymerasecomprising 5′-nuclease activity. As a result, the fluorescentreporter-labeled moiety of the probe labeled with the fluorescentreporter and fluorescence quencher is digested, and the fluorescentreporter is released. Herein, the DNA polymerase with 5′-nucleaseactivity is preferably Taq DNA polymerase, but is not limited thereto.In this method, the released fluorescent reporter is then detected, andthe fluorescent reporter emission is compared with that of a control.

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a probe is synthesized in which a sequencecomplementary to the 3′-flanking nucleotide sequence comprising anucleotide sequence described in any of the above (1) to (28), or anucleotide composing a base pair with the above nucleotide in thecomplementary strand, is combined with an unrelated sequence (step (b)).Then another probe, complementary at the 5′-flanking region of thenucleotide sequence described in any of the above (1) to (28), or thenucleotide composing a base pair with the above nucleotide in thecomplementary strand, is synthesized (step (c)). Then (step (d)), theprobe synthesized in step (c) is hybridized with the DNA prepared instep (a). The DNA hybridized in step (d) is then digested with a singlestrand DNA cleavage enzyme, liberating part of the probe synthesized instep (b) (step (e)). Herein, the single strand DNA cleavage enzyme isnot specifically limited; for example, cleavase can be used as describedbelow. In this method, the probe liberated in step (e) is thenhybridized with a probe for detection (step (f)). Then (step (g)), theDNA hybridized in step (f) is enzymatically digested, and the intensityof fluorescence thus emitted is measured (step (g)). Then (step (h)),the fluorescence intensity measured in step (g) is compared with that ofa control.

An example of the above method is the Invader method (Strategies in SNPgene polymorphism. Kennichi Matsubara and Yoshiyuki Sakaki.Nakayama-Shoten p 94-105; Genome Research 10: 330-343 (2000)).Specifically, a probe complementary to a template in the 3′-flankingregion of a nucleotide sequence described in any of the above (1) to(28), or a nucleotide composing a base pair with the above nucleotide inthe complementary strand, and comprising a sequence unrelated to thetemplate (flap) in the 5′-flanking region, is first synthesized (probeA). Next, a probe comprising a sequence complementary to the template inthe 5′-flanking region of the nucleotide sequence described in any ofthe above (1) to (28), or the nucleotide composing a base pair with theabove nucleotides in the complementary strand, is synthesized (probe B).In probe B, the nucleotide corresponding to a nucleotide described inany of the above (1) to (28), or the nucleotide composing a base pairwith the above nucleotide in the complementary strand, may be anyspecies. Then, the probes are hybridized with the prepared template DNA.The nucleotide in probe B that corresponds to the nucleotide sequencedescribed in any of the above (1) to (28), or the nucleotide composing abase pair with the above nucleotide in the complementary strand, thencreates a region comprising a flap at the 5′-end as a result ofinvasion. Thus, the hybridized DNA is digested with an endonuclease(cleavase) that recognizes the region comprising the flap, and cutsprobe A at the 3′-side of the corresponding nucleotide. As a result, theflap is released. The released flap is then hybridized with a detectionprobe. The detection probe is generally called a fluorescence resonanceenergy transfer (FRET) probe. The probe has a 5′-region in which it canform complementary binding, and the 3′-region is complementary to theflap. Within the 5′-region, which can associate with itselfcomplementarily, the 5′-end and 3′-side thereof are respectively labeledwith the fluorescent reporter and fluorescence quencher. Uponhybridization, the nucleotides in the 3′-end of the released flap invadeinto the complementary binding sites of the FRET probe labeled with thefluorescent reporter, and create a structure that is recognized bycleavase. Herein, the fluorescent reporter released by digestion of theregion labeled with the fluorescent reporter using cleavase is detected,and the intensity of the measured fluorescence is compared with that ofa control.

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a DNA comprising a nucleotide as described in any ofthe above (1) to (28), or a nucleotide composing a base pair with theabove nucleotide in the complementary strand, is amplified (step (b)).The amplified DNA is then dissociated into single-strands (step (c)).Next (step (d)), only one strand of the dissociated single-strand DNAsis isolated. A single nucleotide elongation reaction is then performedfrom close to a nucleotide described in any of the above (1) to (28), ora nucleotide composing a base pair with the above nucleotide in thecomplementary strand; the pyrophosphate thus generated is enzymaticallyilluminated; and the intensity of this illumination is measured (step(e)). Then, the fluorescence intensity measured in step (e) is comparedwith that of a control (step (f)). An example of such a method is thePyrosequencing method (Anal. Biochem. 10: 103-110 (2000)).

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a DNA comprising a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand, is amplified (step (b)). Aprimer is then synthesized that is complementary to the sequence upuntil the nucleotide next to the nucleotide described in any of theabove (1) to (28), or the nucleotide composing a base pair with theabove nucleotide in the complementary strand (step (c)). Then (step(d)), a single nucleotide extension reaction is carried out in thepresence of fluorescently labeled nucleotides, using the DNA amplifiedin step (b) as a template, with the primers synthesized in step (c).Then, fluorescence polarization is measured (step (e)). The fluorescencepolarization measured in step (e) is then compared with that of acontrol (step (f)). An example of such a method is the AcycloPrimemethod (Genome Research 9: 492-498 (1999)).

The AcycloPrime method uses a pair of primers for amplifying the genome,and a single primer for detecting SNPs. First, a genomic regioncontaining SNPs is amplified by PCR. This step is performed as instandard genomic PCR. Next, the amplified PCR product is annealed with aprimer for detecting polymorphisms, and an elongation reaction iscarried out. The primer for detecting polymorphisms is designed toanneal to a region adjacent to the target polymorphic site. Normally,the nucleotide substrate for the elongation reaction is nucleotidederivatives labeled with a fluorescent dye with its 3′-OH blocked(terminator). Therefore, the elongation is stopped on incorporation of asingle nucleotide complementary to a nucleotide at a polymorphic site.Incorporation of the nucleotide derivative into the primer can bedetected as an increase in fluorescence polarization (FP), due to anincrease in molecular weight. By labeling using two different FP dyesthat comprise distinct wavelengths, a nucleotide at a particularpolymorphic site can be distinguished between two nucleotides. Becausethe level of fluorescence polarization can be quantified, it is possibleto determine whether a target allele is homogenous or heterogeneous byrunning a single analysis. The above step (A) in the methods of thepresent invention may be preferably performed by the AcycloPrime method.

One skilled in the art can appropriately prepare the primers for genomicamplification and primers for detecting polymorphisms used in theAcycloPrime method, according to the information on genomic sequence andpolymorphic sites. Examples of the primers for genomic amplification andprimers for detecting polymorphisms, used in the methods of thisinvention to distinguish rice varieties using the AcycloPrime method,are shown in Tables 8 and 9, but they are not limited thereto. TABLE 8Polymorphism Genomic (SNP) PCR detection Primer SEQ SEQ nucleotide SEQMarker Primer nucleotide ID ID Posi- sequence ID Termi- chr cM namesequence (5′-3′) NO: mer Size NO: tion (5′-3′) mer NO: nator SNPs 1 96.1S0015 GCA ATT CCC ACT GGA AGA AT 29 20 803 1 593 AGG TCG ACA CTT CGG 2085 C/T Nipponbare Others TAA GTT GGG GAA TGC GAT GT 30 20 bp CCG TT C T3 20.3 S0040 TCT GCT GCC TCT GCA CAT AC 31 20 902 2 304 GAA CAG CTG TAATAA 21 86 A/T Nipponbare Others AAA AAC GAC ACC ACA TCA GCA 32 21 bp GACTGA T A 3 69.2 S0279 GGG GCG CTC CTT CAA AAC TT 33 20 796 3 450 GAT CCCTGC AAA GTC 20 87 C/T Nipponbare Others GGT TTG GCA CAC CAC AAT GG 34 20bp CCC AC C T 3 146.4 S0044 TGC AAT GTG CCA TTC CAT AG 35 20 901 4 377CGC AAA CCA TCA ACT 20 88 C/T Nipponbare Others TAT GAC AAG GTG GGC CCTAA 36 20 bp TAC AA T C 6 19.1 S0252 CGC CAC AGA ACG GAC AAA AG 37 20 8045 163 CGA TTG GCA GAT AAA 22 89 C/T Nipponbare Others GAC CAA TCC TTTGCC GAA GC 38 20 bp GTT GGA T T C 7 35.7 S0109 CCG ATG GCA GCA CAA ATCTT 39 20 850 6 624 TGG CTA GAA GTA GAT 20 90 C/T Nipponbare Others TCAGTT TGG CTT GGG TGT CC 40 20 bp GCT GC T C 7 84.1 S0115 CCA TTG GTT GGTGTG GCT GT 41 20 784 7 534 AAA CAG GTG AGG GAA 20 91 G/T NipponbareOthers TGG TCG CGG CTG ATA AGC TA 42 20 bp AGA TG T G 7 91.7 S0107 TGCGAT GGA GGG AGT ATT GG 43 20 808 8 358 GAC TGA AAA GTT GTG 20 92 A/GNipponbare Others TGC GAG CGT ACA CCG CTA GT 44 20 bp TGT GT A G 7 99.3S0126 GCT TGA GGC ACG TCA AAA TG 45 20 791 9 475 CAT GAA ATT ATT ACA 2593 G/T Nipponbare Others TTC CGT CGT TCA TGT TGG TC 46 20 bp GAA CTA CAGA T G 7 105.7 S0124 CCC ACG GAA ACA GCC AAA AG 47 20 956 10 323 AGC ACCTCC CCC TCC 20 94 A/G Nipponbare Others TGC TGC CAT GCA AAG AAT CG 48 20bp TCT AA G A 8 20.2 S0146 ATT CGA ACG GGG GAT CCA GT 49 20 859 11 612GGA ACT AGC CCG TGA 20 95 A/C Nipponbare Others AGC GGA TCC TGC TGA TGAGG 50 20 bp CGC TC C A 8 44.6 S0135 GTG CTG CAA AGG GGA GTA TG 51 20 85212 765 GAG AGT CGA GAT GAT 20 96 G/T Nipponbare Others CGC CAA CCT CGTAAA TCA AA 52 20 bp CCA AA G T 9 55.9 S0155 GAA CCT GAG GAC CAA GTG AAAGAG T 53 25 1300 13 571 CAG CTA TAG CCT AGC 20 97 G/T Nipponbare OthersGTA GAG AGG AGA GGG AGA AGG AGG A 54 25 bp TTG GA G T 10 5.5 S0161 ATACCA CAG GTG CTG CGT GA 55 20 340 14 660 GAA GAC AGC TTC TGC 23 98 A/GNipponbare Others TGC GCA ACT AGG GAT TTT CC 56 20 bp TGG TTT GT A G

TABLE 9 Polymorphism Genomic (SNP) PCR detection Primer SEQ SEQnucleotide SEQ Marker Primer nucleotide ID ID Posi- sequence ID Termi-chr cM name sequence (5′-3′) NO: mer Size NO: tion (5′-3′) mer NO: natorSNPs 11 20.3 S0177 CCT TGT GGT CAC ACT TGC GG 57 20 488 15 223 AAC GTCATG GAC GAT 20 99 A/G Nipponbare Others CGG TCT TGA GGT CCA GGG TG 58 20bp CCG CT G A 11 35.6 S0178 TGG CAT CTT TGC ATG TTG AGC 59 21 460 16 247GCC ATG AAA GCA CTG 20 100 C/T Nipponbare Others GCA TCC AGC TGC ACA TTTCC 60 20 bp AAA AA C T 11 80.5 S0174 GAA TCG GTT GCA GGA GAG GG 61 20311 17 163 TTG AGT TCT TGG GGA 20 101 A/G Nipponbare Others GCG GCT ATGCCA TGT TTT TAC C 62 22 bp TTT GT G A 11 85.7 S0185 CGA CCC CAT GAA GCTTTT GC 63 20 644 18 421 TGT TAC AAG CAA AGC 25 102 A/C Nipponbare OthersAAA TCC ACG ACC TCC ACC CCT 64 21 bp ATG AGG AAT G A C 12 42.7 S0208 CTCCCT CCG CTC CCA GAA AT 65 20 500 19 178 AGC TCG AGC TCG AAG 20 103 C/GNipponbare Others ATT TTG GTG GAG CGT CCC CT 66 20 bp ATG GC C G 1 181.8S0007 GCA TGG ATG ACC CTG CTA AT 67 20 802 20 141 CAA ACA TTT AAA ATA 28104 A/G Nipponbare Others TGA TGC CGT TGA CTT TTT GA 68 20 bp TAA ATCATG AAT A A G 5 55.5 S0070 CTT GCT TGG GCA ATC GTC AA 69 20 897 21 480TAA GCC CCC GGC CGA 25 105 A/G Nipponbare Others GTT GCT GAC GCG ACC AGTGT 70 20 bp ACC GGC AAA G A G 8 40.2 S0310 GCT TTG CTT GTT TGA CCA CTC G71 22 802 22 481 GAC TAC AAT CTT CCA 20 106 C/T Nipponbare Others CCATTT TCA TGT CGT GGC TTG 72 21 bp CTC CA T C 4 97.7 S0375 ACA CAA GTG TGCCAT TTT GC 73 20 901 23 131 TGT GAA CTA CAC TAT 26 107 G/C NipponbareOthers TGC CAA GCT ACC TGA GAA CA 74 20 bp TAA GTT GCT TA G C 11 35.6S0346 CGT GCT TGG ATT TTT GTA AGC 75 21 677 24 510 CTG GGA CTT GGA ATG21 108 G/A Nipponbare Others GCA TCC AGC TGC ACA TTT CC 76 20 bp TTT GTTG A 1 161.5 S0013 AAA TTC GGA ATG GCT AGC TG 77 20 798 25 248 GCT AATGTG AAT TAG 22 109 C/T Nipponbare Others ACC TCC GAT GAT TCA ACC AA 7820 bp CCC CCC T C T 11 55.1 S0347 CAA GCG AAG ACT GGA GAG GTT 79 21 29226 92 AGT TTA ACT ATA TAT 28 110 G/C Nipponbare Others ACG TGC TGG CCTCCT ATG TT 80 20 bp AGC ATA CTG ATT C C G 3 94.9 S0330 ATC AAG CAC GATCGG AAA CG 81 20 888 27 743 CAT CTT ATG GTT TAG 23 111 A/G NipponbareOthers ATG GCC GTG GAC TCC AAG TT 82 20 bp GAG GAA TT A G 8 55.4 S0336GAC GAA ATT GTT TCG CCC CTA 83 21 787 28 552 GTC TAT TTG GTA CCA 21 112C/T Nipponbare Others GCC TTC GAG TGG TTT GAC GA 84 20 bp CTT TCT C T

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a DNA comprising a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand, is amplified (step (b)). Then, aprimer that is complementary to a sequence covering up to the nucleotidesequence next to the nucleotide described in any of the above (1) to(28), or the nucleotide composing a base pair with the above nucleotidein the complementary strand, is synthesized (step (c)). Then (step (d)),a single nucleotide elongation reaction is carried out in the presenceof fluorescently labeled nucleotides, using the DNA amplified in step(b) as a template, with the primer synthesized in step (c). Then (step(e)), the nucleotides used in the reaction of step (d) are determinedusing a sequencer. The nucleotide sequence determined in step (e) isthen compared with a control (step (f)). An example of such methods isthe SNuPe method (Rapid Commun. Mass Spectrom. 14: 950-959 (2000)).

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a DNA comprising a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand, is amplified (step (b)). Themolecular weight of the DNA amplified in step (b) is then measured usinga mass spectrometer (step (c)). The molecular weight obtained in step(c) is then compared with that of a control (step (d)). An example ofsuch a method is the MALDI-TOF MS method (Trends Biotechnol. 18: 77-84(2000)).

Furthermore, in another method, DNAs are first prepared from a test rice(step (a)). Then, a DNA comprising a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand, is amplified (step (b)). Then, asubstratum on which a nucleotide probe is immobilized is provided (step(c)).

Herein, “substratum” means a planar material that can immobilizenucleotides. In this invention, nucleotides include oligonucleotides andpolynucleotides. The substratum of this invention is not limited to anyspecific substratum, as long as it allows nucleotide immobilization, butin general, substrata used in DNA array technology may be suitably used.DNA arrays are generally composed of thousands of nucleotides printed ona substratum at high density. Normally, DNAs are printed on thenon-porous surface of a substratum. Generally, the surface of asubstratum is glass, but a porous membrane such as a nitrocellulosemembrane may be used.

In this invention, an example of a method for immobilizing nucleotides(array) is an array developed by Affymetrix, mainly composed ofoligonucleotides. In such an oligonucleotide array, the oligonucleotidesare normally synthesized in situ. For example, methods for in situoligonucleotide synthesis using photolithographic technology(Affymetrix) and inkjet for immobilizing chemical compounds (RosettaInpharmatics) are already known, and any technique can be used toconstruct the substrata of this invention.

The nucleotide probes immobilized on the substratum are not limited toany specific probes, as long as they allow detection of singlenucleotide polymorphisms at a nucleotide described in any of the above(1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand. Thus, the probe may be a probethat can specifically hybridize with a DNA comprising a nucleotidedescribed in any of the above (1) to (28), or a nucleotide composing abase pair with the above nucleotide in the complementary strand, forexample. The nucleotide probe may not be completely complementary to aDNA comprising the nucleotide described in any of the above (1) to (28),or a nucleotide composing a base pair with the above nucleotide in thecomplementary strand, as long as the hybridization is specific. In thisinvention, when an oligonucleotide is immobilized, the length of thenucleotide probe immobilized on a substratum is normally 10 to 100bases, preferably 10 to 50 bases, and more preferably 15 to 25 bases.

In this method, the DNA of step (b) is then contacted with thesubstratum of step (c) (step (d)). This step allows the DNA to hybridizewith the above nucleotide probe. The solutions and conditions forhybridization may vary depending on many factors, including the lengthof the nucleotide probe immobilized on the substratum, but hybridizationcan generally be performed using a method commonly known to one skilledin the art.

In this method, the strength of hybridization between the DNA andnucleotide probe immobilized on the substratum is then detected (step(e)). The detection may be performed by scanning the fluorescence signalon a scanner, for example. In a DNA array, a DNA immobilized on slideglass is generally called a probe, and a labeled DNA in solution iscalled a target. Thus, in the present description, the above nucleotidesimmobilized on the substratum are described as nucleotide probes. Inthis method, the intensity detected in step (e) is then compared withthat of a control (step (f)).

Examples of such methods are methods using DNA arrays (Strategies in SNPgene polymorphism. Kennichi Matsubara and Yoshiyuki Sakaki.Nakayama-Shoten p 128-135; Nature Genetics 22: 164-167 (1999)).

In addition to the above methods, allele-specific oligonucleotide (ASO)hybridization may be used to detect only those mutations at specificpositions. An oligonucleotide comprising a nucleotide sequence in whicha mutation is supposed to exist is prepared, and used for hybridizationwith a DNA. If a mutation is present, the efficiency of hybrid formationis reduced. Such changes can be detected by methods such as Southernblotting, or methods using the properties of special fluorescentreagents that are quenched upon intercalation into gaps within hybrids,or the like.

In addition, the present invention provides oligonucleotides that areprimers for distinguishing between rice varieties, for use in amplifyingDNA regions that comprise a nucleotide described in any of the above (1)to (28), or a nucleotide composing a base pair with an above nucleotidein the complementary strand. An example of such an oligonucleotide maybe an oligonucleotide designed such that it spans a nucleotide describedin any of the above (1) to (28), or a nucleotide composing a base pairwith the above nucleotide in the complementary strand. PCR primers maybe designed and synthesized by methods commonly known to those skilledin the art. The PCR primers are not limited in length, and are normally15 to 100 bp, and preferably 17 to 30 bp. The present invention alsoprovides oligonucleotides that comprise a nucleotide sequencecomplementary to a sequence covering up to a nucleotide next to anucleotide described in any of the above (1) to (28), or a nucleotidecomposing a base pair with the above nucleotide in the complementarystrand. Such an oligonucleotide is useful as a primer for use in themethods of this invention, for distinguishing rice varieties by theAcycloPrime method, for example. Examples of such oligonucleotides arethose shown in Table 8 or 9.

Furthermore, the present invention provides oligonucleotides comprisingat least 15 nucleotides, for use in the methods for distinguishingbetween rice varieties, which can hybridize with a DNA region comprisinga nucleotide described in any of the above (1) to (28), or a nucleotidecomposing a base pair with the above nucleotide in the complementarystrand. The oligonucleotides can be used as a probe, for example.

Such oligonucleotides can hybridize specifically with a DNA regioncomprising a nucleotide described in any of the above (1) to (28), or anucleotide composing a base pair with the above nucleotide in thecomplementary strand. Here, to “hybridize specifically” means that theoligonucleotide does not generate a significant amount ofcross-hybridization with other DNAs under standard hybridizationconditions, and preferably under stringent conditions (for example, theconditions described in Sambrook et al. Molecular Cloning. Cold SpringHarbor Laboratory Press, New York, U.S.A. second edition, (1989)). Aslong as hybridization is specific, an oligonucleotide does not need tobe completely complementary to a DNA region comprising a nucleotide asdescribed in any of the above (1) to (28), or a nucleotide composing abase pair with the above nucleotide in the complementary strand. Thelength of the oligonucleotide is not limited, as long as it is 15nucleotides or longer. Such oligonucleotides can be prepared using acommercial oligonucleotide synthesizer, for example. Alternatively, theymay be prepared as double-stranded DNA fragments obtained by restrictionenzyme treatment and the like.

In addition, the oligonucleotides to be used are preferablyappropriately labeled. The methods for labeling may include a method inwhich the 5′-end of the oligonucleotide is labeled with ³²P byphosphorylating with T4 polynucleotide kinase, a method in whichnucleotide substrates labeled with an isotope such as ³²P, a fluorescentdye, biotin, or the like are incorporated into the oligonucleotidesusing a primer such as random hexamer oligonucleotides and a DNApolymerase such as Klenow enzyme (the random primer method).Furthermore, the present invention also includes oligonucleotides of 15nucleotides or longer that comprise a polymorphic mutation, according toany of the above (1′) to (28′), in a nucleotide described in any of theabove (1) to (28), or a nucleotide composing a base pair with the abovenucleotide in the complementary strand.

Furthermore, the present invention provides kits for distinguishing ricevarieties, comprising the above oligonucleotides of the invention. Thekits of this invention may further comprise an alkaline aqueoussolution. The kits may be also packaged with a standard rice sample foruse as a control, instructions describing the method for using the kit,and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of SEQ ID NO: 1, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 2 shows the nucleotide sequence of SEQ ID NO: 2, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 3 shows the nucleotide sequence of SEQ ID NO: 3, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 4 shows the nucleotide sequence of SEQ ID NO: 4, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 5 shows the nucleotide sequence of SEQ ID NO: 5, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 6 shows the nucleotide sequence of SEQ ID NO: 6, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 7 shows the nucleotide sequence of SEQ ID NO: 7, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 8 shows the nucleotide sequence of SEQ ID NO: 8, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 9 shows the nucleotide sequence of SEQ ID NO: 9, which indicates asite polymorphic between 24 rice varieties, as well as the sequences ofprimers for amplifying a DNA region comprising that site.

FIG. 10 shows the nucleotide sequence of SEQ ID NO: 10, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 11 shows the nucleotide sequence of SEQ ID NO: 11, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 12 shows the nucleotide sequence of SEQ ID NO: 12, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 13 shows the nucleotide sequence of SEQ ID NO: 13, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 14 shows the nucleotide sequence of SEQ ID NO: 14, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 15 shows the nucleotide sequence of SEQ ID NO: 15, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 16 shows the nucleotide sequence of SEQ ID NO: 16, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 17 shows the nucleotide sequence of SEQ ID NO: 17, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 18 shows the nucleotide sequence of SEQ ID NO: 18, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 19 shows the nucleotide sequence of SEQ ID NO: 19, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 20 shows the nucleotide sequence of SEQ ID NO: 20, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 21 shows the nucleotide sequence of SEQ ID NO: 21, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 22 shows the nucleotide sequence of SEQ ID NO: 22, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 23 shows the nucleotide sequence of SEQ ID NO: 23, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 24 shows the nucleotide sequence of SEQ ID NO: 24, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 25 shows the nucleotide sequence of SEQ ID NO: 25, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 26 shows the nucleotide sequence of SEQ ID NO: 26, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 27 shows the nucleotide sequence of SEQ ID NO: 27, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 28 shows the nucleotide sequence of SEQ ID NO: 28, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 29 shows the nucleotide sequence of SEQ ID NO: 29, which indicatesa site polymorphic between 24 rice varieties, as well as the sequencesof primers for amplifying a DNA region comprising that site.

FIG. 30 shows a photograph showing the results of PCR using DNAextracted from polished rice as a template. The polished rice sample wasa commercial rice, said to be “Akitakomachi produced in IbarakiPrefecture in Heisei 12 (the year 2000)”. The primers used for the PCRwere PGC1001 (U: 5′-accgggtagggaaacaaaac-3′/SEQ ID NO: 113; L:5′-aataatacttcggcgcatcg-3′/SEQ ID NO: 114). PCR was carried out usingDNA extracted by the methods below as a template, and the reactionmixture was separated by electrophoresis on a 1.5% agarose gel.

M: molecular weight marker (φX/HaeIII);

1: Method 1 (CTAB);

2: Method 2 (alkali+CTAB);

3: Method 3 (simple extraction);

4: Method 4 (simple extraction+phenol:chloroform treatment);

5: Method 5 (alkali+simple extraction);

6: Method 6 (alkali+simple extraction+phenol:chloroform treatment);

7: control (DNA extracted from a green leaf of Habataki by CTAB, 40 ng);and

8: control (DNA extracted from a green leaf of Sasanishiki by CTAB, 40ng).

BEST MODE FOR CARRYING OUT THE INVENTION

This invention will be explained in detail below with reference toExamples, but it is not to be construed as being limited thereto.

EXAMPLE 1 Detection of Single Nucleotide Polymorphisms (SNPs)

Primers for amplifying 800 bp to 1 kbp of rice genomic DNA were designedusing publicly available rice genome analysis information on the RiceGenome Research Program homepage (http://rgp.dna.affrc.go.jp/), and ricegenomic sequences registered in DDBJ (http://www.ddbj.nig.ac.jp/).Regions not predicted to comprise genes were mainly used for thechromosomal regions with publicly available rice genomic nucleotidesequences, and RFLP marker probe sequences and the like were used forregions other than these. The primer design support site, Primer3(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), was usedto design the primers.

Using the designed primers, first, PCR amplification was performed usingAmpli Taq Gold (Applied Biosystems) and DNA extracted by a simple methodfrom rice varieties Nipponbare, Koshihikari, Kasalath, Guang-lu-ai 4(G4, below), Kitaake, and a wild rice (Oryza rufipogon, W1943) as atemplate. To confirm the amplified fragments, a portion of the reactionmixture was separated by electrophoresis on an agarose gel. The rest ofthe reaction mixture was treated with ExoSAP-IT (Amersham Biosciences)to remove unreacted primers and dNTPs, and then subjected to asequencing reaction as a template. One of the original primers, used forthe first amplification, was again added to the template, and samplesfor sequencing were prepared by performing a cycle sequencing reactionusing the DYEnamic ET Dye Terminator Cycle Sequencing kit for MegaBACE(Amersham Biosciences). Sequencing was carried out using the MegaBACE1000 DNA Sequencing System (Molecular Dynamics). The obtained sequencedata was compared between the varieties to search for single nucleotidesubstitution polymorphisms. Sequencing was performed at least twice foreach variety with each primer, and only certain cases were considered tobe polymorphisms.

Sites showing single nucleotide polymorphisms between Nipponbare andKoshihikari, and between Nipponbare and Kitaake, were further examinedby similarly performing PCR and sequencing using genomic DNA extractedby a simple method from Nipponbare, Hatsushimo, Mutsuhomare, Yukinosei,Kirara 397, Tsugaruroman, Gohyakumangoku, Morinokumasan, Yumeakari,hanaechizen, Koshihikari, Tsukinohikari, Akitakomachi, Asanohikari,Aichinokaori, Matsuribare, Hinohikari, Yumetsukushi, Hitomebore,Manamusume, Fusaotome, Dontokoi, Kinuhikari, and Sasanishiki astemplates, and comparing the nucleotide sequences at the polymorphicsites for each of the varieties. FIGS. 1 to 28 show the polymorphismsfound among the above 24 rice varieties. Polymorphic data are shownaccording to the following rules:

[Rules for Data Description]

(1) Primer sites are indicated by brackets, and the upper primer siteand lower primer site are marked with “p:” and “q:”, respectively.

Example: actctactta a[p:gcagagcga tgaacctgca] atattgagaa aactc[q:aatcacgccc atccttgcct]

(2) SNP positions are shown by brackets and an identification number.

Example: cg[1a]agag[2aa]cttc[3a[4c4]cattt gggg[5c5]acac3]c

Note: In general, identification numbers were attached to both thebeginning and ending brackets; however, the number might be omitted fromthe latter bracket where the correspondence was obvious.

(3) The analyzed variety is indicated by a code below the attachedsequence. Variety codes are separated by “/”.

Example: nhb/ksh/kal/gla/pwl/kta

[Variety code] Each of the above rice varieties is indicated by anabbreviation using three alphabet letters. For example, Nipponbare andKoshihikari are “nhb” and “ksh”, respectively.

(4) The variety code was followed by SNP data, shown as “identificationnumber, variety code: SNP”.

Example: 1 ksh:g

OTHER EXAMPLES

(5) Deletions are indicated by “−”. Regardless of the number of deletednucleotides, only one “−” was used.

Example: g[5agg]ggtcat ctgttacatt atag

5kal:−

(6) Where deletions occurred in the same position but varied in lengthdepending on the variety:

Example: gtttg[20a:gtat[20b:t ccattatgta ttatttcatt tgct20b]t20a]ttatg

20akal:−, 20bgla:−

Since the deletion occurs in the same position, the same identificationnumber was used. However, differences in deletion length were clarifiedby alphabet letters to differentiate, such as “20a:” and “20b:”.

(7) For insertions, “−” was inserted in the published sequence. A single“−” was used.

Example: tacaca[7−]gtca attttattca

7kal:aa

Next, primers for detecting SNPs were designed for those SNPs useful indistinguishing the varieties, and a single nucleotide terminatorreaction was performed using the AcycloPrime-FP kit (Perkin Elmer), toprepare samples for genotyping. Genotyping was performed by measuringfluorescence polarization with ARVO (Perkin Elmer).

The results showed that the markers generated at those positionsdetermined by sequencing to be SNPs showed distinct patterns among thevarieties, and could be used in combination for variety classification(Tables 2 to 7). Tables 8 and 9 show data for the generated SNP markers,such as primer sequences and the SNP sites used.

EXAMPLE 2 Examination of the Methods for DNA Extraction From PolishedRice, Unpolished Rice, and Cooked Rice

Methods for extracting DNA from polished, unpolished, and cooked ricewere examined. First, a single kernel of polished, unpolished, andcooked rice was placed into a 2 ml tube (Eppendorf), and 0.4 ml ofextraction buffer (1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, 0.1 N NaOH) andzirconia balls of 3 mm in diameter were added thereto. The tubes werestood with their lid on for 30 minutes at 4° C. The kernels weredisrupted using a Retch disrupter mixer mill MM300 for two rounds at 300Hz for two minutes, and a milky solution was obtained. The solution wascentrifuged at 10,000 rpm for ten minutes, and the resulting supernatant(0.3 ml) was transferred to a fresh tube. After the addition of 0.3 mlof isopropanol, the solution was well mixed and centrifuged again at10,000 rpm for ten minutes. The supernatant was discarded, and 1 ml of70% ethanol was added to the pellet, and then centrifuged at 10,000 rpmfor three minutes. The supernatant was discarded, and the pellet wasdried and then dissolved in 30 μl of sterilized water (Method 5).

Alternatively, after transferring the supernatant (0.3 ml) to a freshtube in Method 5, 0.3 ml of phenol:chloroform (1:1) was added, and thesolution was mixed well and centrifuged at 10,000 rpm for ten minutes.The supernatant was then transferred to a fresh tube, and processed forisopropanol precipitation (Method 6).

Alternatively, the composition of the extraction buffer used in Methods5 and 6 was changed to 1 M KCl, 10 mM Tris-HCl, and 1 mM EDTA (Methods 3and 4, respectively).

In other alternative methods, CTAB extraction was used. Specifically, apolished rice kernel and 0.2 ml of CTAB buffer (Method 1), or 0.2 ml of0.1 N NaOH (Method 2) were put in a 2 ml tube, and zirconia balls of 3mm in diameter were added thereto. With the tube top closed, the kernelwas disrupted under the same conditions as for Method 5. 0.7 ml of CTABbuffer was then added, and heated at 56° C. for 20 minutes. 640 μl ofphenol:chloroform (1:1) was added to the solution, which was then mixed,and then centrifuged at 10,000 rpm for ten minutes. The supernatant (0.7ml) was transferred to a fresh tube, and 1.3 ml of CTAB precipitationbuffer was added. This was then centrifuged at 10,000 rpm for tenminutes. The pellet was dissolved by adding 0.5 ml of 1 N NaClcontaining RNase, then 1 ml ethanol was added, mixed, and centrifuged at10,000 rpm for ten minutes. The pellet was washed with 1 ml of 70%ethanol, dried, and dissolved in 30 μl of sterilized water.

The DNAs obtained by the above methods were used as templates for PCRusing the primers PGC1001 (U: 5′-accgggtagggaaacaaaac-3′/SEQ ID NO: 113;L: 5′-aataatacttcggcgcatcg-3′/SEQ ID NO: 114).

These results are shown in FIG. 30. While no amplified product wasobtained using DNA extracted from polished rice by Method 1 or 2, goodamplification was observed with DNA extracted using Methods 3 to 6. Thisaccordingly indicates that phenol:chloroform treatment is unnecessarywhen extracting DNA from polished rice, and thus that Methods 3 or 5 arethe simplest methods. The difference between Methods 3 and 5 lay in thebuffer used to disrupt the kernels, which was alkaline in Method 5. Analkaline buffer is advantageous in that the polished rice tissues arequickly rendered fragile, and satisfactory disruption is easilyachieved. Thus, Method 5 was chosen as the simplest and most efficientmethod.

For unpolished rice and cooked rice, an amplified product was notobtained for DNA extracted by Methods 1 and 2, although amplificationwas observed for that of Methods 3 to 6. The best amplification wasobserved for DNA extracted by Method 6. Thus, a method using alkalinebuffer and phenol:chloroform treatment was shown most effective forextracting DNA from unpolished or cooked rice.

EXAMPLE 3 Distinguishing Varieties of Polished Rice

Commercial polished rice indicated to be “100% Akitakomachi produced inIbaraki Prefecture in Heisei 12 (the year 2000)” was purchased. 32kernels were randomly selected, and DNA was separately extracted fromevery single kernel using Method 5. PCR was carried out using theextracted DNAs as templates, and primers for the three markers (S0115,S0146, and S0178) necessary and sufficient to distinguish Akitakomachifrom the other 25 rice varieties. Furthermore, AcycloPrime reactionswere performed using the PCR products as templates, and the singlenucleotide polymorphisms were determined.

As a result, 27 kernels were identified as Akitakomachi, but threekernels turned out to be varieties other than Akitakomachi. Two of thesekernels were not distinguished since one of the three markers did notgive a result. Based on their patterns, the three kernels determined notto be Akitakomachi were presumably either Kirara 397, Koshihikari,Yumetsukushi, or Kinuhikari.

The above results confirmed that the present invention could be used todistinguish between varieties of polished rice.

EXAMPLE 4 Identification of Varieties of Polished Rice

In order to determine the variety of the three kernels which weredetermined in Example 3 to not be Akitakomachi, and which might beKirara 397, Koshihikari, Yumetsukushi, or Kinuhikari, PCR was performedusing the extracted DNAs as templates and primers for the two markers(S0015, S0045) required and sufficient to distinguish between the threevarieties. Furthermore, AcycloPrime reactions were performed using thePCR products as templates, and the single nucleotide polymorphisms weredetermined.

The results showed that all three of the kernels had the same pattern asKoshihikari. Therefore, it was presumed that the polished rice used inExample 3 very likely contained Koshihikari in addition to Akitakomachi.

EXAMPLE 5 Inspection of the Blending Ratio of Polished Rice

A polished rice said to be “Kirara 397, 30%; Tsugaruroman, 40%;Hitomebore, 30%” was inspected to determine whether the three varietieswere blended as indicated. 32 kernels were randomly selected from thepolished rice, and DNA was separately extracted from every kernel usingMethod 5. PCR was performed using the extracted DNAs as templates, andprimers for the seven markers (S0115, S0135, S0161, S0252, S0310, S0336,and S0375) necessary and sufficient to distinguish Kirara 397,Tsugaruroman, and Hitomebore from among the 26 rice varieties that canbe distinguished. Furthermore, AcycloPrime reactions were performedusing the PCR products as templates, and the single nucleotidepolymorphisms were determined.

The results indicated that seven kernels were from Kirara 397, elevenwere from Tsugaruroman, and five were from Hitomebore, while two kernelswere not from any of these three varieties. The other seven kernels werenot determined since some of the seven markers did not provide data.According to the ratio of the three varieties based on the 25 kernelsfor which data could be collected, the blending ratio of the inspectedpolished rice was presumed to be Kirara 397, 28%; Tsugaruroman, 44%; andHitomebore, 20%; with other varieties being 4%.

INDUSTRIAL APPLICABILITY

The present invention provides methods for distinguishing between ricevarieties. Traditional methods for distinguishing varieties based ontheir cultivation traits require inspection by the eyes of experiencedbreeders, and thus simple distinctions are difficult. Furthermore, thevariety of every rice kernel could not be distinguished. In contrast,the methods of this invention examine polymorphisms in the rice genome,and thus enable varieties to be accurately distinguished using a minuteamount of rice sample. Furthermore, the methods of this invention can beapplied to accurately distinguish between closely related varieties.

1. A method of distinguishing between rice varieties, comprising thefollowing steps (a) and (b): (a) determining the type of a nucleotide ata position according to any of the following (1) to (28) in the ricegenome, or a nucleotide on the complementary strand that composes a basepair with the nucleotide at the position: (1) position 593 in thenucleotide sequence of SEQ ID NO: 1, (2) position 304 in the nucleotidesequence of SEQ ID NO: 2, (3) position 450 in the nucleotide sequence ofSEQ ID NO: 3, (4) position 377 in the nucleotide sequence of SEQ ID NO:4, (5) position 163 in the nucleotide sequence of SEQ ID NO: 5, (6)position 164 in the nucleotide sequence of SEQ ID NO: 6, (7) position534 in the nucleotide sequence of SEQ ID NO: 7, (8) position 358 in thenucleotide sequence of SEQ ID NO: 8, (9) position 475 in the nucleotidesequence of SEQ ID NO: 9, (10) position 323 in the nucleotide sequenceof SEQ ID NO: 10, (11) position 612 in the nucleotide sequence of SEQ IDNO: 11, (12) position 765 in the nucleotide sequence of SEQ ID NO: 12,(13) position 571 in the nucleotide sequence of SEQ ID NO: 13, (14)position 660 in the nucleotide sequence of SEQ ID NO: 14, (15) position223 in the nucleotide sequence of SEQ ID NO: 15, (16) position 247 inthe nucleotide sequence of SEQ ID NO: 16, (17) position 163 in thenucleotide sequence of SEQ ID NO: 17, (18) position 421 in thenucleotide sequence of SEQ ID NO: 18, (19) position 178 in thenucleotide sequence of SEQ ID NO: 19, (20) position 141 in thenucleotide sequence of SEQ ID NO: 20, (22) position 480 in thenucleotide sequence of SEQ ID NO: 21, (22) position 481 in thenucleotide sequence of SEQ ID NO: 22, (23) position 131 in thenucleotide sequence of SEQ ID NO: 23, (24) position 510 in thenucleotide sequence of SEQ ID NO: 24, (25) position 248 in thenucleotide sequence of SEQ ID NO: 25, (26) position 92 in the nucleotidesequence of SEQ ID NO: 26, (27) position 743 in the nucleotide sequenceof SEQ ID NO: 27, and (28) position 552 in the nucleotide sequence ofSEQ ID NO: 28, and (b) relating the type of the nucleotide determined instep (a) to a variety of rice.
 2. The method of claim 1, whichdistinguishes the type of a nucleotide by using a polymorphic markercharacterized by a mutation of any of the following (1) to (28) in therice genome: (1) the nucleotide at position 593 in the nucleotidesequence of SEQ ID NO: 1 is T, (2) the nucleotide at position 304 in thenucleotide sequence of SEQ ID NO: 2 is T, (3) the nucleotide at position450 in the nucleotide sequence of SEQ ID NO: 3 is A, (4) the nucleotideat position 377 in the nucleotide sequence of SEQ ID NO: 4 is C, (5) thenucleotide at position 163 in the nucleotide sequence of SEQ ID NO: 5 isC, (6) the nucleotide at position 624 in the nucleotide sequence of SEQID NO: 6 is C, (7) the nucleotide at position 534 in the nucleotidesequence of SEQ ID NO: 7 is C, (8) the nucleotide at position 358 in thenucleotide sequence of SEQ ID NO: 8 is G, (9) the nucleotide at position475 in the nucleotide sequence of SEQ ID NO: 9 is G, (10) the nucleotideat position 323 in the nucleotide sequence of SEQ ID NO: 10 is A, (11)the nucleotide at position 612 in the nucleotide sequence of SEQ ID NO:11 is A, (12) the nucleotide at position 765 in the nucleotide sequenceof SEQ ID NO: 12 is T, (13) the nucleotide at position 571 in thenucleotide sequence of SEQ ID NO: 13 is T, (14) the nucleotide atposition 660 in the nucleotide sequence of SEQ ID NO: 14 is G, (15) thenucleotide at position 223 in the nucleotide sequence of SEQ ID NO: 15is A, (16) the nucleotide at position 247 in the nucleotide sequence ofSEQ ID NO: 16 is A, (17) the nucleotide at position 163 in thenucleotide sequence of SEQ ID NO: 17 is A, (18) the nucleotide atposition 421 in the nucleotide sequence of SEQ ID NO: 18 is C, (19) thenucleotide at position 178 in the nucleotide sequence of SEQ ID NO: 19is G, (20) the nucleotide at position 141 in the nucleotide sequence ofSEQ ID NO: 20 is G, (21) the nucleotide at position 480 in thenucleotide sequence of SEQ ID NO: 21 is C, (22) the nucleotide atposition 481 in the nucleotide sequence of SEQ ID NO: 22 is C, (23) thenucleotide at position 131 in the nucleotide sequence of SEQ ID NO: 23is C, (24) the nucleotide at position 510 in the nucleotide sequence ofSEQ ID NO: 24 is A, (25) the nucleotide at position 248 in thenucleotide sequence of SEQ ID NO: 25 is T, (26) the nucleotide atposition 92 in the nucleotide sequence of SEQ ID NO: 26 is C, (27) thenucleotide at position 743 in the nucleotide sequence of SEQ ID NO: 27is G, and (28) the nucleotide at position 552 in the nucleotide sequenceof SEQ ID NO: 28 is T.
 3. The method of claim 1 or comprising thefollowing steps (a) to (c): (a) preparing DNA from a test rice, (b)amplifying a DNA comprising a nucleotide in a position of any of (1) to(28) of claim 1, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, and (c) determining thenucleotide sequence of the amplified DNA.
 4. The method of claim 1 Icomprising the following steps (a) to (d): (a) preparing DNA from a testrice, (b) digesting the prepared DNA with a restriction enzyme, (c)fractionating the DNA fragments by size, and (d) comparing the size ofthe detected DNA fragment with a control.
 5. The method of claim 1,comprising the following steps (a) to (e): (a) preparing DNA from a testrice, (b) amplifying a DNA comprising a nucleotide in a position of anyof (1) to (28) of claim 1, or a nucleotide in the complementary strandcomposing a base pair with the nucleotide at the position, (c) digestingthe amplified DNA with a restriction enzyme, (d) fractionating the DNAfragments by size, and (e) comparing the size of the detected DNAfragment with a control.
 6. The method of claim 1 or comprising thefollowing steps (a) to (e): (a) preparing DNA from a test rice, (b)amplifying a DNA comprising a nucleotide in a position of any of (1) to(28) of claim 1, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, (c) denaturing theamplified DNA into single-stranded DNA, (d) fractionating the denaturedsingle-stranded DNA on a non-denaturing gel, and (e) comparing themobility of the fractionated single-stranded DNA on the gel with acontrol.
 7. The method of claim 1 o, comprising the following steps (a)to (f): (a) preparing DNA from a test rice, (b) synthesizing twodifferent oligonucleotide probes labeled with a reporter fluorescencedye and quencher fluorescence dye, where an oligonucleotide iscomplementary to a proximal nucleotide sequence comprising a nucleotidein a position of any of (1) to (28) of claim 1, or a nucleotide in thecomplementary strand composing a base pair with the nucleotide at theposition, (c) hybridizing the DNA prepared in step (a) with the probesynthesized in step (b), (d) amplifying a DNA comprising a nucleotide ina position of any of (1) to (28) of claim 1, or a nucleotide in thecomplementary strand composing a base pair with the nucleotide at theposition, (e) detecting the emission of reporter fluorescence, and (f)comparing the emission of reporter fluorescence detected in step (e)with a control.
 8. The method of claim 1 I; comprising the followingsteps (a) to (h): (a) preparing DNA from a test rice, (b) synthesizing aprobe in which a sequence complementary to the 3′-flanking nucleotidesequence comprising a nucleotide in a position of any of (1) to (28) ofclaim 1, or a nucleotide in the complementary strand composing a basepair with the nucleotide at the position, is combined with a totallyunrelated sequence, (c) synthesizing a probe that is complementary tothe 5′-flanking region comprising a nucleotide in a position of any of(1) to (28) of claim 1, or a nucleotide in the complementary strandcomposing a base pair with the nucleotide at the position, (d)hybridizing the probe synthesized in step (c) with the DNA prepared instep (a), (e) digesting the hybridized DNA in step (d) with asingle-stranded DNA cleaving enzyme, and dissociating a part of theprobe synthesized in step (b), (f) hybridizing the dissociated probe instep (e) with a probe for detection, (g) enzymatically digesting thehybridized DNA in step (f), and measuring the fluorescence intensitythus generated, and (h) comparing the fluorescence intensity measured instep (g) with a control.
 9. The method of claim 1 o, comprising thefollowing steps (a) to (f): (a) preparing DNA from a test rice, (b)amplifying a DNA comprising a nucleotide in a position of any of (1) to(28) of claim 1, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, (c) denaturing theamplified DNA into single-stranded DNAs, (d) separating only one strandfrom the denatured single-stranded DNAs, (e) performing an elongationreaction from near a position of any of (1) to (28) of claim 1, or anucleotide in the complementary strand composing a base pair with thenucleotide at the position, whereby the reaction elongates onenucleotide at a time, then enzymatically illuminating the generatedpyrophosphate, and measuring the intensity of the illumination, and (f)comparing the fluorescence intensity measured in step (e) with acontrol.
 10. The method of claim 1, comprising the following steps (a)to (f): (a) preparing DNA from a test rice, (b) amplifying a DNAcomprising a nucleotide in a position of any of (1) to (28) of claim 1,or a nucleotide in the complementary strand composing a base pair withthe nucleotide at the position, (c) synthesizing a probe complementaryto a nucleotide sequence comprising a sequence covering up to anucleotide adjacent to a position of any of (1) to (28) of claim 1, or anucleotide in the complementary strand composing a base pair with thenucleotide at the position, (d) performing a single nucleotide extensionreaction in the presence of fluorescently labeled nucleotides, using theDNA amplified in step (b) as a template, and the primer synthesized instep (c), (e) measuring the fluorescence polarization, and (f) comparingthe fluorescence polarization measured in step (e) with a control. 11.The method of claim 1 o, comprising the following steps (a) to (f): (a)preparing DNA from a test rice, (b) amplifying a DNA comprising anucleotide in a position of any of (1) to (28) of claim 1, or anucleotide in the complementary strand composing a base pair with thenucleotide at the position, (c) synthesizing a primer complementary to anucleotide sequence comprising a sequence covering up to the nucleotideadjacent to a position of any of (1) to (28) of claim 1, or a nucleotidein the complementary strand composing a base pair with the nucleotide atthe position, (d) performing a single nucleotide extension reaction inthe presence of fluorescently labeled nucleotides, using the DNAamplified in step (b) as a template, and the primer synthesized in step(c), (e) determining the nucleotide variety used in the reaction of step(d) using a sequencer, and (f) comparing the nucleotide determined instep (e) with a control.
 12. The method of claim 1 o, comprising thefollowing steps (a) to (d): (a) preparing DNA from a test rice, (b)amplifying a DNA comprising a nucleotide in a position of any of (1) to(28) of claim 1, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, (c) measuring themolecular weight of the DNA amplified in step (b) using a massspectrometer, and (d) comparing the molecular weight measured in step(c) with a control.
 13. The method of claim 1 o, comprising thefollowing steps (a) to (f): (a) preparing DNA from a test rice, (b)amplifying a DNA comprising a nucleotide in a position of any of (1) to(28) of claim 1, or a nucleotide in the complementary strand composing abase pair with the nucleotide at the position, (c) providing asubstratum on which a nucleotide probe is immobilized, (d) contactingthe DNA of step (b) with the substratum of step (c), (e) detecting thestrength of hybridization between the DNA and the nucleotide probeimmobilized on the substratum, and (f) comparing the strength detectedin step (e) with a control.
 14. The method of claim 1, furthercomprising the following steps (a) and (b): (a) disrupting a rice seedin an alkaline aqueous solvent, and (b) extracting rice genomic DNA fromthe seed disrupted in step (a).
 15. The method of claim 14, wherein therice seed is polished.
 16. A primer for distinguishing between ricevarieties, wherein the primer is (a) an oligonucleotide foramplification of a DNA region comprising a nucleotide in a position ofany of (1) to (28) of claim 1 in the rice genome, or a nucleotide in thecomplementary strand composing a base pair with the nucleotide at theposition, or (b) an oligonucleotide comprising a nucleotide sequencecomplementary to a sequence covering up to a nucleotide adjacent to aposition of any of (1) to (28) of claim 1 in the rice genome, or anucleotide in the complementary strand composing a base pair with thenucleotide at the position.
 17. An oligonucleotide for distinguishingbetween rice varieties, wherein the oligonucleotide hybridizes with aDNA region comprising a nucleotide in a position of any of (1) to (28)of claim 1, or a nucleotide in the complementary strand composing a basepair with the nucleotide at the position, comprising at least 15nucleotides.
 18. A kit for distinguishing between rice varieties,comprising the oligonucleotide of claim
 17. 19. The kit of claim 18,further comprising an alkaline aqueous solvent.
 20. A kit fordistinguishing between rice varieties, comprising the primer of claim16.
 21. The kit of claim 20, further comprising an alkaline aqueoussolvent.